Combination methods for treating cancers

ABSTRACT

Disclosed are methods for treating a cancer in a subject (e.g., a human) in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula I: 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt thereof, in combination with a vinca-alkaloid, or a pharmaceutically acceptable salt thereof. The subject may be very high risk or high risk for the cancer and may not respond to either agent administered as a sole therapy. The subject who has the cancer may also be refractory to at least one chemotherapy treatment, or is in relapse after treatment with chemotherapy, or both. The cancer may be a hematologic malignancy, such as leukemia or lymphoma, or a solid tumor cancer, such as pancreatic, lung and colon cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/796,795, filed on Jul. 10, 2015, now U.S. Pat. No. 9,707,236, whichclaims priority to and the benefit of U.S. Provisional Application No.62/024,424, filed on Jul. 14, 2014, which is hereby incorporated byreference in its entirety.

FIELD

The present disclosure relates generally to the field of therapeuticsand medicinal chemistry, and more specifically to the use of certainSpleen Tyrosine Kinase (Syk) inhibitors in combination with the use ofcertain vinca-alkaloids in the treatment of cancer including, forexample, leukemia, lymphoma and solid-cell tumors.

BACKGROUND

A number of imidazopyrazine compounds are under investigation forinhibiting Spleen Tyrosine Kinase (Syk) activity. Syk is a non-receptortyrosine kinase that plays critical roles in immunoreceptor- andintegrin-mediated signaling in a variety of cell types, includingB-cells, macrophages, monocytes, mast cells, eosinophils, basophils,neutrophils, dendritic cells, T-cells, natural killer cells, platelets,and osteoclasts.

Syk has been reported to play an important role in signaling through theB-cell receptor, known to be an important survival signal in B-cells. Assuch, inhibition of Syk activity may be useful for treating certaintypes of hematologic malignancies. Examples of such hematologicmalignancies include cancer, such as B-cell lymphoma and leukemia.Furthermore, there are reports of Syk expression in certain solid cancer(tumor) cell lines. Examples of such solid cancer tumors includepancreatic cancer, lung cancer, colon and colo-rectal cancer, ovariancancer and hepatocellular cancer. Additionally, the inhibition of Sykactivity is believed to be useful for treating of other diseases andconditions, including inflammatory diseases (e.g., rheumatoidarthritis), allergic disorders and autoimmune diseases.

One such compound that has been found to inhibit Syk activity isrepresented by formula I:

or a pharmaceutically acceptable salt thereof. The compound of formulahas the chemical name6-(1H-indazol-6-yl)-N-(4-morpholinophenyl)imidazo[1,2-a]pyrazin-8-amine.This compound and its synthesis have been described in U.S. Pat. Nos.8,450,321 and 8,455,493, which are hereby incorporated by reference intheir entirety and specifically with respect to the method of makingthis compound. See e.g., U.S. Pat. No. 8,450,321, Examples 1 and 2.

Vinca-alkaloids are a subset of drugs derived from the Madagascarperiwinkle plant that were discovered in the 1950's, and have a varietyof uses from treating diabetes, high blood pressure, and cancer. Thereare approximately 10 (ten) vinca-alkaloids either currently in use or indevelopment for these indications, including the four majorvinca-alkaloids in clinical oncology use: vinblastine, vinorelbine,vincristine, and vindesine. Each of these major vinca-alkaloids has beenreported to cause serious side effects, most notably neuropathy. One ofthe more commonly known vinca-alkaloids is vincristine (VCR), also knownas leurocristine and marketed as Oncovin. As with other vinca-alkaloids,vincristine is useful in cancer chemotherapy as a mitotic inhibitor andis commonly used in the standard of care regimen CHOP (Cyclophosphamide,Hydroxydaunorubicin (also known as doxyrubicin), Oncovin (vincristine)and Prednisone) for non-Hodgkin's disease or R-CHOP (CHOP in combinationwith Rituxan® (also known as rituximab) for B-cell lymphomas. However,vincristine also shares several serious side effects with the othervinca-alkaloids, the most serious of which is chemotherapy-inducedperipheral neuropathy, a progressive, enduring, often irreversibleneuropathy. This neuropathy can be so severe as to result in thereduction or even cessation of use of vincristine.

What is desired are methods for treating diseases responsive to theinhibition of Syk in subjects in need of such treatment, including insubjects who may be considered at risk for the disease, are refractoryto standard treatments, and/or are in relapse after standard treatments,wherein a treatment regimen of a Syk inhibitor alone does not result ininhibition of cell activity, especially for subjects who may besensitive to neuropathy secondary to standard dosing levels ofvincristine or vinca-alkaloid containing treatment regimens.

SUMMARY

Provided herein are methods for treating cancer in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of a compound of formula I:

or a pharmaceutically acceptable salt thereof, in combination with avinca-alkaloid, or a pharmaceutically acceptable salt thereof.

In some embodiments, the subject is a human who has a cancer responsiveto Syk activity. In another embodiment, the subject is a human who has asolid cancer tumor which expresses Syk. In some embodiments, the subjectis a human who has a 17p deletion, a TP53 mutation, NOTCH1, a SF3B1mutation, a 11q deletion, or any combination thereof. In one embodiment,the subject is a human who has a 17p deletion, a TP53 mutation, or acombination thereof. In another embodiment, the subject is a human whohas NOTCH1, a SF3B1 mutation, a 11q deletion, or any combinationthereof.

In some embodiments, the vinca-alkaloid is selected from the groupconsisting of vincristine, vinblastine, vindesine, vinorelbine,desoxyvincaminol, vincaminol, vinburnine, vincamajine, and vineridine,and pharmaceutically acceptable salts thereof. In certain embodiments,at least one vinca-alkaloid is selected from the group consisting ofvincristine, vinblastine, vindesine, vinorelbine, desoxyvincaminol,vincaminol, vinburnine, vincamajine, and vineridine and pharmaceuticallyacceptable salts thereof. In some embodiments, the vinca-alkaloid isselected from the group consisting of vincristine, vinblastine,vindesine, and vinorelbine, and pharmaceutically acceptable saltsthereof. In other embodiments, the vinca-alkaloid is selected from thegroup consisting of vincristine and vinblastine, and pharmaceuticallyacceptable salts thereof. In one embodiment, the vinca-alkaloid isvincristine and pharmaceutically acceptable salts thereof. In anotherembodiment, the vinca-alkaloid is vinblastine and pharmaceuticallyacceptable salts thereof.

Provided herein are also methods for treating cancer in a subject inneed thereof, comprising administering to the subject a therapeuticallyeffective amount of a compound of formula I, or a pharmaceuticallyacceptable salt thereof, and a therapeutically effective amount of avinca-alkaloid, or a pharmaceutically acceptable salt thereof.

Provided herein are also methods for treating cancer in a subject inneed thereof, comprising administering to the subject a therapeuticallyeffective amount of a compound of formula I, or a pharmaceuticallyacceptable salt thereof, and a therapeutically effective amount of avinca-alkaloid, or a pharmaceutically acceptable salt thereof, wherein:the subject is a human who is not undergoing any other anti-cancertreatments; and the subject is (i) refractory to at least oneanti-cancer treatment, or (ii) in relapse after treatment with at leastone anti-cancer therapy, or a combination thereof.

Provided herein are also figures and examples illustrating that thecombination of the compound of formula I, or a pharmaceuticallyacceptable salt thereof, and a therapeutically effective amount of avinca-alkaloid, or a pharmaceutically acceptable salt thereto, hasunexpected improvements over the effects of the compound of formula I,or the vinca-alkaloid, alone in monotherapy or administered as a soleagent in the treatment of certain cancers and their respective celllines.

In some embodiments, the subject is not undergoing any other anti-cancertreatments using one or more PI3K inhibitors. Such PI3K inhibitors mayinclude, in certain embodiments, Compounds A, B and C, whose structuresare provided below.

Compound A has the chemical name(S)-2-(1-((9H-purin-6-yl)amino)propyl)-5-fluoro-3-phenylquinazolin-4(3H)-one,Compound B is named(S)-2-(1-((9H-purin-6-yl)amino)ethyl)-5-fluoro-3-phenylquinazolin-4(3H)-one,and Compound C is(S)-2-(1-((9H-purin-6-yl)amino)ethyl)-3-(2,6-difluorophenyl)quinazolin-4(3H)-one.

In some embodiments, the subject is refractory to at least oneanti-cancer treatment. In other embodiments, the subject is in relapseafter treatment with at least one anti-cancer treatment.

In some embodiments, about 100 mg to 800 mg of the compound of formulaI, or a pharmaceutically acceptable salt thereof, is administered tosubject twice daily. In other embodiments, about 200 mg to 400 mg of thecompound of formula I, or a pharmaceutically acceptable salt thereof, isadministered to the subject twice daily. In one embodiment, about 400 mgof the compound of formula I, or a pharmaceutically acceptable saltthereof, is administered to subject twice daily.

In one variation, the subject is a human who has a 17p deletion, a TP53mutation, or a combination thereof; and about 100 mg to 800 mg of thecompound of formula I, or a pharmaceutically acceptable salt thereof, isadministered to the subject twice daily. In another variation, thesubject is a human who has a 17p deletion, a TP53 mutation, or acombination thereof; and about 200 mg to 400 mg of the compound offormula I, or a pharmaceutically acceptable salt thereof, isadministered to the subject twice daily. In yet another variation, thesubject is a human who has a 17p deletion, a TP53 mutation, or acombination thereof; and about 400 mg of the compound of formula I, or apharmaceutically acceptable salt thereof, is administered to the subjecttwice daily.

In other embodiments, the vinca-alkaloid, or a pharmaceuticallyacceptable salt thereof, is administered to the subject once a week atclinically approved or sub-clinically approved amounts. In someembodiments, the amount of the vinca-alkaloid is administered to thesubject once a week at a sub-clinically approved amount. In otherembodiments, the vinca-alkaloid is vincristine and the amount ofvincristine, or a pharmaceutically acceptable salt thereof, isadministered at a dose between 0.1 mg-M² and 1.5 mg-M². In otherembodiments, the vinca-alkaloid is administered to the subject once aweek at a dose of between 0.25 mg-M2 and 1.0 mg-M2 and thevinca-alkaloid is selected from the group consisting of vincristine andvinblastine. In other embodiments, the vinca-alkaloid is administered tothe subject once daily at a dose of between 0.1 mg-M2 and 0.2 mg-M2 andthe vinca-alkaloid is selected from the group consisting of vincristineand vinblastine.

In certain embodiments, the compound of formula (I) or apharmaceutically acceptable salt thereof is administered before thevinca-alkaloid, or a pharmaceutically acceptable salt thereof. In otherembodiments, the vinca alkaloid, or a pharmaceutically acceptable saltthereof, is administered before the compound of formula (I) or apharmaceutically acceptable salt thereof.

In one embodiment, the compound of formula (I) or a pharmaceuticallyacceptable salt thereof, and the vinca-alkaloid, or a pharmaceuticallyacceptable salt thereof, are administered simultaneously, wherein thevinca-alkaloid is administered via IV and the compound of formula I isadministered via tablet. In certain embodiments, the compound of formulaI and the at least one vinca-alkaloid, or pharmaceutically acceptablesalts thereof, is independently administered twice a day. In otherembodiments, the compound of formula (I) or a pharmaceuticallyacceptable salt thereof, and the vinca-alkaloid, or a pharmaceuticallyacceptable salt thereof, are administered once a day. In otherembodiments, the compound of formula (I) or a pharmaceuticallyacceptable salt thereof, and the vinca-alkaloid, or a pharmaceuticallyacceptable salt thereof, are administered once a week. In oneembodiment, the compound of formula (I) or a pharmaceutically acceptablesalt thereof, is administered twice a day, and the vinca-alkaloid, or apharmaceutically acceptable salt thereof, is administered once a week.In one embodiment, the compound of formula (I) or a pharmaceuticallyacceptable salt thereof, is administered once a day, and thevinca-alkaloid, or a pharmaceutically acceptable salt thereof, isadministered once a week

In some embodiments, the cancer is a hematologic malignancy. In certainembodiments, the cancer is a leukemia. In one embodiment, the leukemiais chronic lymphocytic leukemia (CLL). In certain embodiments, thecancer is a lymphoma. In one embodiment, the lymphoma is non-Hodgkin'slymphoma (NHL). In one variation, the NHL is diffuse large B-celllymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL),small lymphocytic lymphoma (SLL), lymphoplasmacytic lymphoma (LPL),and/or marginal zone lymphoma (MZL). Thus, it is understood that in oneaspect the subject is a human who has a hematologic malignancy, such asleukemia or lymphoma.

In certain embodiments, the cancer is selected from the group consistingof acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML),chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),myelodysplastic syndrome (MDS), myeloproliferative disease (MPD),chronic myeloid leukemia (CIVIL), multiple myeloma (MM), non-Hodgkin'slymphoma (NHL), indolent non-Hodgkin's lymphoma (iNHL), refractory iNHL,mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldestrom'smacroglobulinemia (WM), T-cell lymphoma, B-cell lymphoma, diffuse largeB-cell lymphoma (DLBCL), lymphoplasmacytic lymphoma (LPL), and marginalzone lymphoma (MZL).

In some embodiments, the cancer is a solid tumor cancer (or solid cancertumor). In certain embodiments the cancer is a solid tumor and expressesspleen tyrosine kinase (Syk) activity. In other embodiments, the solidtumor cancer is selected from the group consisting of pancreatic, lung,colorectal cancer, ovarian, breast, esophageal, adenocarcinoma andhepatocellular.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts the chemical structure of the compound of Formula I.

FIG. 2A details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe DHL-2 cell line.

FIG. 2B details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe DHL-10 cell line.

FIG. 2C details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe DHL-4 cell line.

FIG. 2D details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe Karpas-422 cell line.

FIG. 2E details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe DHL-6 cell line.

FIG. 2F details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe OCI-LY3 cell line.

FIG. 2G details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe OCI-LY4 cell line.

FIG. 2H details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe OCI-LY19 cell line.

FIG. 2I details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe OCI-LY7 cell line.

FIG. 2J details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe Pfeiffer cell line.

FIG. 2K details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe KMS-11 cell line.

FIG. 2L details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe RPMI-8226 cell line.

FIG. 2M details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe OPM2 cell line.

FIG. 2N details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe MM1.S2 cell line.

FIG. 2O details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe WSU-FSCCL cell line.

FIG. 2P details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe WSU-NHL cell line.

FIG. 2Q details the inhibition effects on the combination of thecompound of formula I and vincristine compared to vincristine alone inthe Mino cell line.

FIG. 3A summarizes the effect of the combination of the compound ofFormula I and taxol versus the combination of the compound of Formula Iand combretastatin A4 in the DHL-10 cell line.

FIG. 3B summarizes the effect of the combination of the compound ofFormula I and vincristine versus the combination of the compound ofFormula I and colchicine in the DHL-10 cell line.

FIG. 3C summarizes the effect doxorubicin versus the combination of thecompound of Formula I and doxorubicin in the DHL-10 cell line.

FIG. 4 depicts and summarizes the inhibitory effects of the combinationof a compound of FIG. 1 and one of two of the vinca-alkaloids,vincristine and vinblastine respectively, in the DLBCL cell line, DHL-10when compounds were co-administered.

FIG. 5A summarizes the inhibition of cell viability by the combinationof a compound of FIG. 1 and vincristine in the Syk-expressing malignantpancreatic cell line, MiaPaca.

FIG. 5B summarizes the inhibition of cell viability by the combinationof a compound of FIG. 1 and vincristine in the non-Syk expressingmalignant colon cell line, HepG2.

FIG. 6 depicts the level of Syk expression in the MiaPaca and HepG2malignant colon cell lines.

FIG. 7 summarizes the levels of Syk expression in malignant cell linesfrom lung, pancreas, and colon.

DETAILED DESCRIPTION

The following description sets forth exemplary compositions and methods.It should be recognized, however, that such description is not intendedas a limitation on the scope of the present disclosure but is insteadprovided as a description of exemplary embodiments.

Provided herein are methods for treating cancer in a certain populationof subjects (e.g., humans) in need thereof, comprising administering tosuch subjects a therapeutically effective amount of a compound offormula I:

or a pharmaceutically acceptable salt, in combination with avinca-alkaloid, or a pharmaceutically acceptable salt thereof.

Provided herein are also figures and examples illustrating that thecombination of the compound of formula I, or a pharmaceuticallyacceptable salt thereof, and a therapeutically effective amount of avinca-alkaloid, or a pharmaceutically acceptable salt thereto, hasunexpected improvements over the effects of the compound of formula I,or the vinca-alkaloid, alone in monotherapy or administered as a soleagent in the treatment of certain cancers and their respective celllines.

The compound of formula I may also be referred to by its compound name:6-(1H-indazol-6-yl)-N-(4-morpholinophenyl)imidazo[1,2-a]pyrazin-8-amine.The compound name provided is named using ChemBioDraw Ultra 12.0, andone skilled in the art understands that the compound structure may benamed or identified using other commonly recognized nomenclature systemsand symbols including CAS and IUPAC. One method for synthesizing thecompound of formula I has been previously described in, for example,U.S. Pat. No. 8,450,321.

Any formula or structure given herein, including the compound of formulaI and pharmaceutically acceptable salts thereof, is also intended torepresent unlabeled forms as well as isotopically labeled forms of thecompounds, or salts thereof. Isotopically labeled compounds or saltsthereof have structures depicted by the formulas given herein exceptthat one or more atoms are replaced by an atom having a selected atomicmass or mass number. Examples of isotopes that can be incorporated intocompounds of the disclosure include isotopes of hydrogen, carbon,nitrogen, oxygen, phosphorous, fluorine and chlorine, such as, but notlimited to ²H (deuterium, D), ³H (tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F,³¹P, ³²P, ³⁵S, ³⁶Cl and ¹²⁵I. Various isotopically labeled compounds orsalts thereof of the present disclosure, for example those into whichradioactive isotopes such as ³H, ¹³C and ¹⁴C are incorporated. Suchisotopically labeled compounds or salts thereof may be useful inmetabolic studies, reaction kinetic studies, detection or imagingtechniques, such as positron emission tomography (PET) or single-photonemission computed tomography (SPECT) including drug or substrate tissuedistribution assays or in radioactive treatment of subjects (e.g.humans).

The disclosure also includes the compound of formula I andpharmaceutically acceptable salts thereof, in which from 1 to nhydrogens attached to a carbon atom is/are replaced by deuterium, inwhich n is the number of hydrogens in the molecule. Such compounds mayexhibit increased resistance to metabolism and are thus useful forincreasing the half-life of the compound of formula I, orpharmaceutically acceptable salts thereof when administered to a mammal.See, for example, Foster, “Deuterium Isotope Effects in Studies of DrugMetabolism”, Trends Pharmacol. Sci. 5(12):524-527 (1984). Such compoundsare synthesized by means well known in the art, for example by employingstarting materials in which one or more hydrogens have been replaced bydeuterium.

Deuterium labeled or substituted therapeutic compounds of the disclosuremay have improved DMPK (drug metabolism and pharmacokinetics)properties, relating to distribution, metabolism and excretion (ADME).Substitution with heavier isotopes such as deuterium may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life, reduced dosage requirements and/oran improvement in therapeutic index. An ¹⁸F labeled compound may beuseful for PET or SPECT studies. Isotopically labeled compounds of thisdisclosure and prodrugs thereof can generally be prepared by carryingout the procedures disclosed in the schemes or in the examples andpreparations described below by substituting a readily availableisotopically labeled reagent for a non-isotopically labeled reagent. Itis understood that deuterium in this context is regarded as asubstituent in the compound of formula I and pharmaceutically acceptablesalts thereof.

The concentration of such a heavier isotope, specifically deuterium, maybe defined by an isotopic enrichment factor. In the compounds of thisdisclosure any atom not specifically designated as a particular isotopeis meant to represent any stable isotope of that atom. Unless otherwisestated, when a position is designated specifically as “H” or “hydrogen”,the position is understood to have hydrogen at its natural abundanceisotopic composition. Accordingly, in the compounds or salts thereof ofthis disclosure any atom specifically designated as a deuterium (D) ismeant to represent deuterium.

Vinca alkaloids, and pharmaceutically acceptable salts thereof, arederived from the Madagascar periwinkle plant, and have been used totreat diabetes, high blood pressure, and various cancers. Examples ofvinca-alkaloids include vincristine, vinblastine, vindesine,vinorelbine, desoxyvincaminol, vincaminol, vinburnine, vincamajine, andvineridine. Typically, there have been four major vinca alkaloids inclinical use: vinblastine, vinorelbine, vincristine, and vindesine. Allvinca alkaloids are administered intravenously (IV).

The vinca-alkaloids, and pharmaceutically acceptable salts thereof, ofthe present disclosure are cytotoxics—they halt the division of cellsand cause cell death. During cell division, vinca alkaloid moleculesbind to the building blocks of a protein called tubulin, inhibiting itsformation. Tubulin protein normally works in cells to createmicrotubules. These microtubules provide cells with both the structureand flexibility they need to divide and replicate. Without microtubules,cells cannot divide. As opposed to a Syk inhibitor, which inhibitsspleen tyrosine kinase, vinca-alkaloids mechanism occupying tubulin'sbuilding block structure, thus preventing, in theory, the formation ofmicrotubules and thus interfering with cancer cells' ability to divide.

One of the vinca-alkaloids of this disclosure, vinblastine, inhibitsangiogenesis, or the process by which new blood vessels grow frompre-existing ones. Angiogenesis is an essential step in a tumor'stransition to malignancy. Vinblastine is generally applied to treatHodgkin's disease, non-Hodgkin's lymphoma, breast cancer, and germ celltumors. Side effects of vinblastine include: toxicity to white bloodcells, nausea, vomiting, constipation, dyspnea, chest or tumor pain,wheezing, and fever. Vinblastine is also occasionally associated withantidiuretic hormone secretion and angina.

Another vinca alkaloid of this disclosure is vinorelbine, which issimilar in its effects to vinblastine. Vinorelbine has exhibitedsignificant antitumor activity in patients with breast cancer andantiproliferation effects on osteosarcoma (bone tumor cells).Vinorelbine treatment can result in side effects including decreasedresistance to infection, bruising or bleeding, anemia, constipation,diarrhea, nausea, numbness or tingling in the hands and feet, fatigue(also called peripheral neuropathy), and inflammation at the injectionsite. Less common side effects include hair loss and allergic reaction.

Another example or embodiment of the vinca alkaloids of this disclosureis vincristine, or pharmaceutically acceptable salts thereof.Vincristine has a high affinity for tubulin dimers (dimers are buildingblocks of a protein only two blocks long) and can attach and reattach atdifferent sites quickly, thus in theory preventing the dimers' abilityto reassemble (build) the tubules, thus destabilizing the tubulin andinhibiting microtubule formation. Vincristine is FDA approved to treatacute leukemia, rhabdomyosarcoma, neuroblastoma, Wilm's tumor, Hodgkin'sdisease, and other lymphomas. Vincristine's most common side effectsare: peripheral neuropathy, suppression of bone marrow activity,constipation, nervous system toxicity, nausea, and vomiting, withneuropathy being the most common and serious side effect. As a result,there are reports of some subjects being treated with vincristine foroncology having had to stop vincristine treatment.

The fourth common vinca-alkaloid is vindesine, or pharmaceuticallyacceptable salts thereof. Vindesine has a serum half-life of only 24hours, but similar effects (intended and side) to that of vinblastine.Vindesine is commonly administered at a dose of 3 milligrams per squaremeter of body surface during treatment for melanoma, lung cancers, and(combined with other drugs) uterine cancers. Additional side effectsfrom vindesine include: anemia, blood cell toxicity, fatigue, tinglingor pricking sensations in the skin, and skin toxicity

Pharmaceutically Acceptable Salts

In some embodiments of the methods described herein, a pharmaceuticallyacceptable salt of the compound of formula I is administered to thesubject (e.g., a human).

As used herein, by “pharmaceutically acceptable” refers to a materialthat is not biologically or otherwise undesirable, e.g., the materialmay be incorporated into a pharmaceutical composition administered to apatient without causing any significant undesirable biological effectsor interacting in a deleterious manner with any of the other componentsof the composition in which it is contained. Pharmaceutically acceptablevehicles (e.g., carriers, adjuvants, and/or other excipients) havepreferably met the required standards of toxicological and manufacturingtesting and/or are included on the Inactive Ingredient Guide prepared bythe U.S. Food and Drug administration.

“Pharmaceutically acceptable salts” include, for example, salts withinorganic acids and salts with an organic acid. Examples of salts mayinclude hydrochlorate, phosphate, diphosphate, hydrobromate, sulfate,sulfinate, nitrate, malate, maleate, fumarate, tartrate, succinate,citrate, acetate, lactate, mesylate, p-toluenesulfonate,2-hydroxyethylsulfonate, benzoate, salicylate, stearate, and alkanoate(such as acetate, HOOC—(CH₂)_(n)—COOH where n is 0-4). In addition, ifthe compounds described herein are obtained as an acid addition salt,the free base can be obtained by basifying a solution of the acid salt.Conversely, if the product is a free base, an addition salt,particularly a pharmaceutically acceptable addition salt, may beproduced by dissolving the free base in a suitable organic solvent andtreating the solution with an acid, in accordance with conventionalprocedures for preparing acid addition salts from base compounds. Thoseskilled in the art will recognize various synthetic methodologies thatmay be used to prepare nontoxic pharmaceutically acceptable additionsalts.

The terms “effective amount”, “pharmaceutically effective amount”, and“therapeutically effective amount” refer to an amount that may beeffective to elicit the desired biological or medical response,including the amount of a compound that, when administered to a subjectfor treating a disease, is sufficient to effect such treatment for thedisease. The effective amount will vary depending on the compound, thedisease and its severity and the age, weight, etc., of the subject to betreated. The effective amount can include a range of amounts. Apharmaceutically effective amount includes amounts of an agent which areeffective when combined with other agents.

In some embodiments of the disclosure, the pharmaceutically acceptablesalt of the compound of formula I is a bis-mesylate salt. In someembodiments of the disclosure, the pharmaceutically acceptable salt ofthe vinca-alkaloids is a sulfate salt. In some embodiments of thedisclosure, the pharmaceutically acceptable salt of the compound offormula I is a bis-mesylate salt and the pharmaceutically acceptablesalt of the vinca-alkaloids is a sulfate salt. In one embodiment of thedisclosure, the pharmaceutically acceptable salt of the compound offormula I is a bis-mesylate salt and the vinca-alkaloid is vincristine,wherein the pharmaceutically acceptable salt is vincristine sulfate.

Pharmaceutical Compositions

In some embodiments of the methods described herein, the compound offormula I, or a pharmaceutically acceptable salt, is present in apharmaceutical composition comprising the compound of formula I, or apharmaceutically acceptable salt, and at least one pharmaceuticallyacceptable vehicle. Pharmaceutically acceptable vehicles may includepharmaceutically acceptable carriers, adjuvants and/or other excipients,and other ingredients can be deemed pharmaceutically acceptable insofaras they are compatible with other ingredients of the formulation and notdeleterious to the recipient thereof.

The pharmaceutical compositions of the compound of formula I describedherein can be manufactured using any conventional method, e.g., mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, melt-spinning, spray-drying, or lyophilizingprocesses. An optimal pharmaceutical formulation can be determined byone of skill in the art depending on the route of administration and thedesired dosage. Such formulations can influence the physical state,stability, rate of in vivo release, and rate of in vivo clearance of theadministered agent. Depending on the condition being treated, thesepharmaceutical compositions can be formulated and administeredsystemically or locally.

The term “carrier” refers to diluents, disintegrants, precipitationinhibitors, surfactants, glidants, binders, lubricants, and otherexcipients and vehicles with which the compound is administered.Carriers are generally described herein and also in “Remington'sPharmaceutical Sciences” by E. W. Martin. Examples of carriers include,but are not limited to, aluminum monostearate, aluminum stearate,carboxymethylcellulose, carboxymethylcellulose sodium, crospovidone,glyceryl isostearate, glyceryl monostearate, hydroxyethyl cellulose,hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxyoctacosanylhydroxystearate, hydroxypropyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, lactose, lactose monohydrate, magnesiumstearate, mannitol, microcrystalline cellulose, poloxamer 124, poloxamer181, poloxamer 182, poloxamer 188, poloxamer 237, poloxamer 407,povidone, silicon dioxide, colloidal silicon dioxide, silicone, siliconeadhesive 4102, and silicone emulsion. It should be understood, however,that the carriers selected for the pharmaceutical compositions, and theamounts of such carriers in the composition, may vary depending on themethod of formulation (e.g., dry granulation formulation, soliddispersion formulation).

The term “diluent” generally refers to a substance that are used todilute the compound of interest prior to delivery. Diluents can alsoserve to stabilize compounds. Examples of diluents may include starch,saccharides, disaccharides, sucrose, lactose, polysaccharides,cellulose, cellulose ethers, hydroxypropyl cellulose, sugar alcohols,xylitol, sorbitol, maltitol, microcrystalline cellulose, calcium orsodium carbonate, lactose, lactose monohydrate, dicalcium phosphate,cellulose, compressible sugars, dibasic calcium phosphate dehydrate,mannitol, microcrystalline cellulose, and tribasic calcium phosphate.

The term “disintegrant” generally refers to a substance which, uponaddition to a solid preparation, facilitates its break-up ordisintegration after administration and permits the release of an activeingredient as efficiently as possible to allow for its rapiddissolution. Examples of disintegrants may include maize starch, sodiumstarch glycolate, croscarmellose sodium, crospovidone, microcrystallinecellulose, modified corn starch, sodium carboxymethyl starch, povidone,pregelatinized starch, and alginic acid.

The term “precipitation inhibitors” generally refers to a substance thatprevents or inhibits precipitation of the active agent from asupersaturated solution. One example of a precipitation inhibitorincludes hydroxypropylmethylcellulose (HPMC).

The term “surfactants” generally refers to a substance that lowers thesurface tension between a liquid and a solid that could improve thewetting of the active agent or improve the solubility of the activeagent. Examples of surfactants include poloxamer and sodium laurylsulfate.

The term “glidant” generally refers to substances used in tablet andcapsule formulations to improve flow-properties during tabletcompression and to produce an anti-caking effect. Examples of glidantsmay include colloidal silicon dioxide, talc, fumed silica, starch,starch derivatives, and bentonite.

The term “binder” generally refers to any pharmaceutically acceptablefilm which can be used to bind together the active and inert componentsof the carrier together to maintain cohesive and discrete portions.Examples of binders may include hydroxypropylcellulose,hydroxypropylmethylcellulose, povidone, copovidone, and ethyl cellulose.

The term “lubricant” generally refers to a substance that is added to apowder blend to prevent the compacted powder mass from sticking to theequipment during the tableting or encapsulation process. A lubricant canaid the ejection of the tablet form the dies, and can improve powderflow. Examples of lubricants may include magnesium stearate, stearicacid, silica, fats, calcium stearate, polyethylene glycol, sodiumstearyl fumarate, or talc; and solubilizers such as fatty acidsincluding lauric acid, oleic acid, and C₈/C₁₀ fatty acid.

Methods of Treatment

Provided herein are methods for using a compound of formula I, or apharmaceutically acceptable salt thereof, to selectively or specificallyinhibit Syk activity therapeutically or prophylactically, in combinationwith a vinca-alkaloid, or pharmaceutically acceptable salt thereof, toselectively or specifically inhibit tubulin or microtubule formationtherapeutically or prophylactically. The method comprises administeringa compound of formula I, or a pharmaceutically acceptable salt thereof,or a pharmaceutical composition thereof, in combination with a vincaalkaloid, or a pharmaceutically acceptable salt thereof, to a subject(e.g., a human) in need thereof in an amount sufficient to inhibit Sykactivity and/or inhibit tubulin or microtubule formation. The method canbe employed to treat subjects (e.g., humans) suffering from, or subjectto, a condition whose symptoms or pathology is mediated by Sykexpression or activity.

“Treatment” or “treating” is an approach for obtaining beneficial ordesired results including clinical results. Beneficial or desiredclinical results may include one or more of the following:

-   -   (i) decreasing one more symptoms resulting from the disease;    -   (ii) diminishing the extent of the disease and/or stabilizing        the disease (e.g., delaying the worsening of the disease);    -   (iii) delaying the spread (e.g., metastasis) of the disease;    -   (iv) delaying or slowing the recurrence of the disease and/or        the progression of the disease;    -   (v) ameliorating the disease state and/or providing a remission        (whether partial or total) of the disease and/or decreasing the        dose of one or more other medications required to treat the        disease;    -   (vi) increasing the quality of life, and/or    -   (vii) prolonging survival.

“Delaying” the development of a disease or condition means to defer,hinder, slow, retard, stabilize, and/or postpone development of thedisease or condition. This delay can be of varying lengths of time,depending on the history of the disease or condition, and/or subjectbeing treated. A method that “delays” development of a disease orcondition is a method that reduces probability of disease or conditiondevelopment in a given time frame and/or reduces the extent of thedisease or condition in a given time frame, when compared to not usingthe method. Such comparisons are typically based on clinical studies,using a statistically significant number of subjects. Disease orcondition development can be detectable using standard methods, such asroutine physical exams, mammography, imaging, or biopsy. Development mayalso refer to disease or condition progression that may be initiallyundetectable and includes occurrence, recurrence, and onset.

The compound of formula I, or a pharmaceutically acceptable saltthereof, in combination with a vinca alkaloid, or a pharmaceuticallyacceptable salt thereof, may, in some embodiments, be administered to asubject (e.g., a human) who is at risk or has a family history of thedisease or condition.

The term “inhibition” indicates a decrease in the baseline activity of abiological activity or process. “Inhibition of activity of Syk activity”refers to a decrease in activity of Syk as a direct or indirect responseto the presence of the compound of formula I, or a pharmaceuticallyacceptable salt thereof, relative to the activity of Syk in the absenceof such compound or a pharmaceutically acceptable salt thereof. In someembodiments, the inhibition of Syk activity may be compared in the samesubject prior to treatment, or other subjects not receiving thetreatment. “Inhibition of activity of tubulin formation” refers to adecrease in tubulin formation as a direct or indirect response to thepresence of a vinca-alkaloid, or a pharmaceutically acceptable saltthereof, relative to the activity of tubulin formation in the absence ofsuch vinca-alkaloid or a pharmaceutically acceptable salt thereof. Insome embodiments, the inhibition of tubulin formation may be compared inthe same subject prior to treatment, or other subjects not receiving thetreatment.

Diseases

In some embodiments, the compound of formula I, or a pharmaceuticallyacceptable salt thereof, or a pharmaceutical composition thereof, incombination with a vinca-alkaloid, or a pharmaceutically acceptable saltthereof, is used in the treatment of cancer. In certain embodiments, thecompound of formula I, or a pharmaceutically acceptable salt thereof, incombination with a vinca-alkaloid, or a pharmaceutically acceptable saltthereof, is used in the treatment of a hematologic malignancy. In someembodiments, the compound of formula I, or a pharmaceutically acceptablesalt thereof, in combination with a vinca-alkaloid, or apharmaceutically acceptable salt thereof, inhibits the growth orproliferation of cancer cells of hematopoietic origin. In someembodiments, the cancer cells are of lymphoid origin, and in certainembodiments, the cancer cells are related to or derived from Blymphocytes or B lymphocyte progenitors.

Hematologic malignancies amenable to treatment using the methoddisclosed in the present disclosure include, without limitation,lymphomas (e.g., malignant neoplasms of lymphoid and reticuloendothelialtissues, such as Burkitt's lymphoma, Hodgkins' lymphoma, non-Hodgkins'lymphomas, lymphocytic lymphomas); multiple myelomas; leukemias (e.g.,lymphocytic leukemias, chronic myeloid (myelogenous) leukemias). Othercancer cells, of hematopoietic origin or otherwise, that express Sykalso can be treated by administration of the polymorphs and compositionsthereof described herein.

In particular embodiments, the hematologic malignancy is leukemia orlymphoma. In certain embodiments, the hematologic malignancy is acutelymphocytic leukemia (ALL), acute myeloid leukemia (AML), chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),myelodysplastic syndrome (MDS), myeloproliferative disease (MPD),chronic myeloid leukemia (CML), multiple myeloma (MM), non-Hodgkin'slymphoma (NHL), indolent non-Hodgkin's lymphoma (iNHL), refractory iNHL,mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldestrom'smacroglobulinemia (WM), T-cell lymphoma, B-cell lymphoma, diffuse largeB-cell lymphoma (DLBCL), lymphoplasmacytic lymphoma (LPL), and marginalzone lymphoma (MZL).

In one embodiment, the cancer is T-cell acute lymphoblastic leukemia(T-ALL), or B-cell acute lymphoblastic leukemia (B-ALL). In anotherembodiment, the cancer is chronic lymphocytic leukemia (CLL). In yetanother embodiment, the cancer is non-Hodgkin's lymphoma (NHL). In oneembodiment, the NHL is diffuse large B-cell lymphoma (DLBCL), mantlecell lymphoma (MCL), follicular lymphoma (FL), small lymphocyticlymphoma (SLL), lymphoplasmacytic lymphoma (LPL), and marginal zonelymphoma (MZL). In one embodiment, the cancer is indolent non-Hodgkin'slymphoma (iNHL).

In some embodiments, the compound of formula I, or a pharmaceuticallyacceptable salt thereof, or a pharmaceutical composition thereof, incombination with a vinca-alkaloid, or a pharmaceutically acceptable saltthereof, is used in the treatment of a solid tumor cancer. In certainembodiments, the compound of formula I, or a pharmaceutically acceptablesalt thereof, in combination with a vinca-alkaloid, or apharmaceutically acceptable salt thereof, is used in the treatment ofcertain solid tumor cancers, such as pancreatic cancer, lung cancer,colon cancer, colo-rectal cancer, breast cancer, hepatocellular cancer.In certain embodiments, the compound of formula I, or a pharmaceuticallyacceptable salt thereof, in combination with a vinca-alkaloid, or apharmaceutically acceptable salt thereof, is used in the treatment ofcertain solid tumor cancers which have an expression of Syk activity orin which Syk is expressed. Other solid tumor cancer cells that expressSyk also can be treated by administration of the polymorphs andcompositions thereof described herein.

In yet another aspect, provided are methods of treating a subject (e.g.,a human) having a Syk-mediated disorder by administering a compound offormula I, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition thereof, in combination with avinca-alkaloid, or a pharmaceutically acceptable salt thereof, to thesubject. Provided are also methods of modulating Syk in a subject (e.g.,a human) by administering a compound of formula I, or a pharmaceuticallyacceptable salt thereof, or a pharmaceutical composition thereof, incombination with a vinca-alkaloid, or a pharmaceutically acceptable saltthereof, to the subject.

In any of the methods described herein, the compound of formula I, or apharmaceutically acceptable salt thereof, may be administered to theindividual as a unit dosage, for example in the form of a tablet. In anyof the methods described herein, the vinca-alkaloid, or apharmaceutically acceptable salt thereof, may be administered to theindividual via IV (intravenous) delivery.

Any of the methods of treatment provided herein may be used to treatcancer at an advanced stage. Any of the methods of treatment providedherein may be used to treat cancer at locally advanced stage. Any of themethods of treatment provided herein may be used to treat early stagecancer. Any of the methods of treatment provided herein may be used totreat cancer in remission. In some of the embodiments of any of themethods of treatment provided herein, the cancer has reoccurred afterremission. In some embodiments of any of the methods of treatmentprovided herein, the cancer is progressive cancer.

Subjects

Any of the methods of treatment provided may be used to treat a subjectwho has been diagnosed with or is suspected of having cancer. “Subject”refers to an animal, such as a mammal (including a human), that has beenor will be the object of treatment, observation or experiment. Themethods described herein may be useful in human therapy and/orveterinary applications. In some embodiments, the subject is a mammal.In one embodiment, the subject is a human.

In some of the embodiments of any of the methods provided herein, thesubject is a human who is at risk of developing a cancer (e.g., a humanwho is genetically or otherwise predisposed to developing a cancer) andwho has or has not been diagnosed with the cancer. As used herein, an“at risk” subject is a subject who is at risk of developing cancer(e.g., a hematologic malignancy, or a solid tumor cancer). The subjectmay or may not have detectable disease, and may or may not havedisplayed detectable disease prior to the treatment methods describedherein. An at risk subject may have one or more so-called risk factors,which are measurable parameters that correlate with development ofcancer, such as described herein. A subject having one or more of theserisk factors has a higher probability of developing cancer than anindividual without these risk factor(s).

These risk factors may include, for example, age, sex, race, diet,history of previous disease, presence of precursor disease, genetic(e.g., hereditary) considerations, and environmental exposure. In someembodiments, a subject at risk for cancer includes, for example, asubject whose relatives have experienced this disease, and those whoserisk is determined by analysis of genetic or biochemical markers. Priorhistory of having cancer may also be a risk factor for instances ofcancer recurrence.

Provided herein are methods for treating a subject (e.g., a human) whois at “very high risk” or “high risk” for cancer (e.g., a hematologicmalignancy). Such subjects may be identified by the present of certaingenetic deletions and/or mutations. In one aspect, a very high risksubject is a human who has a 17p deletion, a TP53 mutation, or acombination thereof. In one aspect, a high risk subject is a human whohas NOTCH1, a SF3B1 mutation, a 11q deletion, or any combinationthereof. Thus, it is understood that methods of treatment as detailedherein may, in some instances, employ selecting a subject who is at veryhigh risk or at high risk for cancer by detecting the presence orabsence of one or more 17p deletion, a TP53 mutation, NOTCH1, a SF3B1mutation, a 11q deletion, or any combination thereof.

Provided herein are also methods for treating a subject (e.g., a human)who exhibits one or more symptoms associated with cancer (e.g., ahematologic malignancy or a solid tumor cancer). In some embodiments,the subject is at an early stage of cancer. In other embodiments, thesubject is at an advanced stage of cancer.

Provided herein are also methods for treating a subject (e.g., a human)who is undergoing one or more standard therapies for treating cancer(e.g., a hematologic malignancy or a solid tumor cancer), such aschemotherapy, radiotherapy, immunotherapy, and/or surgery. Thus, in someforegoing embodiments, the compound of formula I, or a pharmaceuticallyacceptable salt thereof, in combination with a vinca-alkaloid, or apharmaceutically acceptable salt thereof, administered before, during,or after administration of chemotherapy, radiotherapy, immunotherapy,and/or surgery.

In certain embodiments, the subject may be a human who is (i) refractoryto at least one anti-cancer therapy, or (ii) in relapse after treatmentwith at least one anti-cancer therapy, or both (i) and (ii). In some ofembodiments, the subject is refractory to at least two, at least three,or at least four anti-cancer therapy (including, for example, standardor experimental chemotherapies).

In certain embodiments, the subject is refractory to at least one, atleast two, at least three, or at least four anti-cancer therapy(including, for example, standard or experimental chemotherapy) selectedfrom fludarabine, rituximab, obinutuzumab, alkylating agents,alemtuzumab and other chemotherapy treatments such as CHOP(cyclophosphamide, doxorubicin, vincristine, prednisone); R-CHOP(rituximab-CHOP); hyperCVAD (hyperfractionated cyclophosphamide,vincristine, doxorubicin, dexamethasone, methotrexate, cytarabine);R-hyperCVAD (rituximab-hyperCVAD); FCM (fludarabine, cyclophosphamide,mitoxantrone); R-FCM (rituximab, fludarabine, cyclophosphamide,mitoxantrone); bortezomib and rituximab; temsirolimus and rituximab;temsirolimus and Velcade®; Iodine-131 tositumomab (Bexxar®) and CHOP;CVP (cyclophosphamide, vincristine, prednisone); R-CVP (rituximab-CVP);ICE (iphosphamide, carboplatin, etoposide); R-ICE (rituximab-ICE); FCR(fludarabine, cyclophosphamide, rituximab); FR (fludarabine, rituximab);and D.T. PACE (dexamethasone, thalidomide, cisplatin, Adriamycin®,cyclophosphamide, etoposide).

Other examples of chemotherapy treatments (including standard orexperimental chemotherapies) are described below. In addition, treatmentof certain lymphomas is reviewed in Cheson, B. D., Leonard, J. P.,“Monoclonal Antibody Therapy for B-Cell Non-Hodgkin's Lymphoma” The NewEngland Journal of Medicine 2008, 359(6), p. 613-626; and Wierda, W. G.,“Current and Investigational Therapies for Patients with CLL” Hematology2006, p. 285-294. Lymphoma incidence patterns in the United States isprofiled in Morton, L. M., et al. “Lymphoma Incidence Patterns by WHOSubtype in the United States, 1992-2001” Blood 2006, 107(1), p. 265-276.

For example, treatment of non-Hodgkin's lymphomas (NHL), especially ofB-cell origin, include the use of monoclonal antibodies, standardchemotherapy approaches (e.g., CHOP, CVP, FCM, MCP, and the like),radioimmunotherapy, and combinations thereof, especially integration ofan antibody therapy with chemotherapy. Examples of unconjugatedmonoclonal antibodies for Non-Hodgkin's lymphoma/B-cell cancers includerituximab, alemtuzumab, human or humanized anti-CD20 antibodies,lumiliximab, anti-TRAIL, bevacizumab, galiximab, epratuzumab, SGN-40,and anti-CD74. Examples of experimental antibody agents used intreatment of Non-Hodgkin's lymphoma/B-cell cancers include ofatumumab,ha20, PRO131921, alemtuzumab, galiximab, SGN-40, CHIR-12.12,epratuzumab, lumiliximab, apolizumab, milatuzumab, and bevacizumab.Examples of standard regimens of chemotherapy for Non-Hodgkin'slymphoma/B-cell cancers include CHOP (cyclophosphamide, doxorubicin,vincristine, prednisone), FCM (fludarabine, cyclophosphamide,mitoxantrone), CVP (cyclophosphamide, vincristine and prednisone), MCP(mitoxantrone, chlorambucil, and prednisolone), R-CHOP (rituximab plusCHOP), R-FCM (rituximab plus FCM), R-CVP (rituximab plus CVP), and R-MCP(R-MCP). Examples of radioimmunotherapy for Non-Hodgkin'slymphoma/B-cell cancers include yttrium-90-labeled ibritumomab tiuxetan,and iodine-131-labeled tositumomab.

In another example, therapeutic treatments for mantle cell lymphoma(MCL) include combination chemotherapies such as CHOP (cyclophosphamide,doxorubicin, vincristine, prednisone), hyperCVAD (hyperfractionatedcyclophosphamide, vincristine, doxorubicin, dexamethasone, methotrexate,cytarabine) and FCM (fludarabine, cyclophosphamide, mitoxantrone). Inaddition, these regimens can be supplemented with the monoclonalantibody rituximab (Rituxan) to form combination therapies R-CHOP,hyperCVAD-R, and R-FCM.

Other approaches include combining any of the abovementioned therapieswith stem cell transplantation or treatment with ICE (iphosphamide,carboplatin and etoposide). Other approaches to treating mantle celllymphoma includes immunotherapy such as using monoclonal antibodies likeRituximab (Rituxan). Rituximab can be used for treating indolent B-cellcancers, including marginal-zone lymphoma, WM, CLL and small lymphocyticlymphoma. A combination of Rituximab and chemotherapy agents isespecially effective. A modified approach is radioimmunotherapy, whereina monoclonal antibody is combined with a radioisotope particle, such asIodine-131 tositumomab (Bexxar®) and Yttrium-90 ibritumomab tiuxetan(Zevalin®). In another example, Bexxar® is used in sequential treatmentwith CHOP. Another immunotherapy example includes using cancer vaccines,which is based upon the genetic makeup of an individual patient's tumor.A lymphoma vaccine example is GTOP-99 (MyVax®). Yet other approaches totreating mantle cell lymphoma includes autologous stem celltransplantation coupled with high-dose chemotherapy, or treating mantlecell lymphoma includes administering proteasome inhibitors, such asVelcade® (bortezomib or PS-341), or antiangiogenesis agents, such asthalidomide, especially in combination with Rituxan. Another treatmentapproach is administering drugs that lead to the degradation of Bcl-2protein and increase cancer cell sensitivity to chemotherapy, such asoblimersen (Genasense) in combination with other chemotherapeuticagents. Another treatment approach includes administering mTORinhibitors, which can lead to inhibition of cell growth and even celldeath; a non-limiting example is Temsirolimus (CCI-779), andTemsirolimus in combination with Rituxan®, Velcade® or otherchemotherapeutic agents.

Other recent therapies for MCL have been disclosed (Nature Reviews;Jares, P. 2007). Such examples include Flavopiridol, PD0332991,R-roscovitine (Selicilib, CYC202), Styryl sulphones, Obatoclax(GX15-070), TRAIL, Anti-TRAIL DR4 and DR5 antibodies, Temsirolimus(CCl-779), Everolimus (RAD001), BMS-345541, Curcumin, Vorinostat (SAHA),Thalidomide, lenalidomide (Revlimid®, CC-5013), and Geldanamycin(17-AAG).

Examples of other therapeutic agents used to treat Waldenstrom'sMacroglobulinemia (WM) include perifosine, bortezomib)(Velcade®,rituximab, sildenafil citrate (Viagra®), CC-5103, thalidomide,epratuzumab (hLL2-anti-CD22 humanized antibody), simvastatin,enzastaurin, campath-1H, dexamethasone, DT PACE, oblimersen,antineoplaston A10, antineoplaston AS2-1, alemtuzumab, beta alethine,cyclophosphamide, doxorubicin hydrochloride, prednisone, vincristinesulfate, fludarabine, filgrastim, melphalan, recombinant interferonalfa, carmustine, cisplatin, cyclophosphamide, cytarabine, etoposide,melphalan, dolastatin 10, indium In 111 monoclonal antibody MN-14,yttrium Y 90 humanized epratuzumab, anti-thymocyte globulin, busulfan,cyclosporine, methotrexate, mycophenolate mofetil, therapeuticallogeneic lymphocytes, Yttrium Y 90 ibritumomab tiuxetan, sirolimus,tacrolimus, carboplatin, thiotepa, paclitaxel, aldesleukin, recombinantinterferon alfa, docetaxel, ifosfamide, mesna, recombinantinterleukin-12, recombinant interleukin-11, Bcl-2 family proteininhibitor ABT-263, denileukin diftitox, tanespimycin, everolimus,pegfilgrastim, vorinostat, alvocidib, recombinant flt3 ligand,recombinant human thrombopoietin, lymphokine-activated killer cells,amifostine trihydrate, aminocamptothecin, irinotecan hydrochloride,caspofungin acetate, clofarabine, epoetin alfa, nelarabine, pentostatin,sargramostim, vinorelbine ditartrate, WT-1 analog peptide vaccine, WT1126-134 peptide vaccine, fenretinide, ixabepilone, oxaliplatin,monoclonal antibody CD19, monoclonal antibody CD20, omega-3 fatty acids,mitoxantrone hydrochloride, octreotide acetate, tositumomab and iodineI-131 tositumomab, motexafin gadolinium, arsenic trioxide, tipifarnib,autologous human tumor-derived HSPPC-96, veltuzumab, bryostatin 1, andPEGylated liposomal doxorubicin hydrochloride, and any combinationthereof.

Examples of therapeutic procedures used to treat WM include peripheralblood stem cell transplantation, autologous hematopoietic stem celltransplantation, autologous bone marrow transplantation, antibodytherapy, biological therapy, enzyme inhibitor therapy, total bodyirradiation, infusion of stem cells, bone marrow ablation with stem cellsupport, in vitro-treated peripheral blood stem cell transplantation,umbilical cord blood transplantation, immunoenzyme technique,pharmacological study, low-LET cobalt-60 gamma ray therapy, bleomycin,conventional surgery, radiation therapy, and nonmyeloablative allogeneichematopoietic stem cell transplantation.

Examples of other therapeutic agents used to treat diffuse large B-celllymphoma (DLBCL) drug therapies (Blood 2005 Abramson, J.) includecyclophosphamide, doxorubicin, vincristine, prednisone, anti-CD20monoclonal antibodies, etoposide, bleomycin, many of the agents listedfor Waldenstrom's, and any combination thereof, such as ICE and R-ICE.

Examples of other therapeutic agents used to treat chronic lymphocyticleukemia (CLL) (Spectrum, 2006, Fernandes, D.) include Chlorambucil(Leukeran), Cyclophosphamide (Cyloxan, Endoxan, Endoxana, Cyclostin),Fludarabine (Fludara), Pentstatin (Nipent), Cladribine (Leustarin),Doxorubicin (Adriamycin®, Adriblastine), Vincristine (Oncovin),Prednisone, Prednisolone, Alemtuzumab (Campath, MabCampath), many of theagents listed for Waldenstrom's, and combination chemotherapy andchemoimmunotherapy, including the common combination regimen: CVP(cyclophosphamide, vincristine, prednisone); R-CVP (rituximab-CVP); ICE(iphosphamide, carboplatin, etoposide); R-ICE (rituximab-ICE); FCR(fludarabine, cyclophosphamide, rituximab); and FR (fludarabine,rituximab).

In yet another aspect, provided is a method of sensitizing a subject(e.g., a human) who is (i) refractory to at least one chemotherapytreatment, or (ii) in relapse after treatment with chemotherapy, or both(i) and (ii), wherein the method comprises administering a compound offormula I, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition thereof, in combination with avinca-alkaloid, or a pharmaceutically acceptable salt thereof, to thesubject. A subject who is sensitized is a subject who is responsive tothe treatment involving administration of the compound of formula I, ora pharmaceutically acceptable salt thereof, in combination with avinca-alkaloid, or a pharmaceutically acceptable salt thereof, to thesubject or who has not developed resistance to such treatment. In oneembodiment of the disclosure, a subject who is sensitized is a subjectwho is responsive to the treatment involving administration of thecompound of formula I, or a pharmaceutically acceptable salt thereof tothe subject or who has not developed resistance to such treatment.

Monotherapy and Combination Therapies

Monotherapy

In one aspect, the compound of formula I, or a pharmaceuticallyacceptable salt thereof, in combination with a vinca-alkaloid, or apharmaceutically acceptable salt thereof, is administered as amonotherapy (i.e. the only treatment regimen) to the subject (e.g., ahuman). Provided herein are methods of treatment in which the compoundof formula I, or a pharmaceutically acceptable salt thereof, incombination with a vinca-alkaloid, or a pharmaceutically acceptable saltthereof, administered to a subject (e.g., a human) is the onlyanti-cancer therapy regimen administered to the subject. Provided hereinare methods of treatment in which the compound of formula I, or apharmaceutically acceptable salt thereof, in combination with avinca-alkaloid, or a pharmaceutically acceptable salt thereof,administered to a subject (e.g., a human), wherein the subject is notundergoing any other anti-cancer treatments. In one variation, thesubject is not undergoing any other anti-cancer treatments using one ormore PI3K inhibitors. Such PI3K inhibitors may include, in certainembodiments, Compounds A, B and C, whose structures are provided below.

In one variation, the subject is not undergoing any other anti-cancertreatments using Compound A, or a pharmaceutically acceptable saltthereof. In another variation, the subject is not undergoing any otheranti-cancer treatments using Compound B, or a pharmaceuticallyacceptable salt thereof. In yet another variation, the subject is notundergoing any other anti-cancer treatments using Compound C, or apharmaceutically acceptable salt thereof.

In some embodiments where the compound of formula I, or apharmaceutically acceptable salt thereof, in combination with avinca-alkaloid, or a pharmaceutically acceptable salt thereof, isadministered as a monotherapy treatment regimen to the subject, thesubject may be a human who is (i) refractory to at least one anti-cancertherapy, or (ii) in relapse after treatment with at least oneanti-cancer therapy, or both (i) and (ii). In some of embodiments, thesubject is refractory to at least two, at least three, or at least fouranti-cancer therapy (including, for example, standard or experimentalchemotherapies). For example, in certain embodiments, the subject may bea human who is (i) refractory to a therapy using an anti-CD20 antibody,an alkylating agent (e.g., bendamustine), a purine analog (e.g.,fludarabine), an anthracycline, or any combination thereof (ii) inrelapse after treatment with an anti-CD20 antibody, an alkylating agent(e.g., bendamustine), a purine analog (e.g., fludarabine), ananthracycline, or any combination thereof, or both (i) and (ii).

A human subject who is refractory to at least one anti-cancer therapyand/or is in relapse after treatment with at least one anti-cancertherapy, as described above, may have undergone one or more priortherapies. In some embodiments, such subjects have undergone one, two,three, or four, or at least one, at least two, at least three, at leastfour, or at least five, or between one and ten, between one and nine,between one and eight, between one and seven, between one and six,between one and five, or between one and four, anti-cancer therapiesprior to treatment using the methods described herein (e.g., prior tothe administration of the compound of formula I, or a pharmaceuticallyacceptable salt thereof, as a monotherapy).

It should be understood that when a subject (e.g. a human) is treatedwith the compound of formula I, or a pharmaceutically acceptable saltthereof, in combination with a vinca-alkaloid, or a pharmaceuticallyacceptable salt thereof, as a monotherapy treatment regimen as describedby this disclosure, the subject may also undergo one or more othertherapies that are not anti-cancer therapies.

In some embodiments, there is provided a method for treating cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a compound of formula I, or apharmaceutically acceptable salt, and a therapeutically effective amountof a vinca-alkaloid, or a pharmaceutically acceptable salt, wherein: thevinca-alkaloid is selected from the group consisting of vincristine,vindesine, vinorelbine and vinblastine, and the subject is a human whois (i) refractory to at least one anti-cancer treatment, or (ii) inrelapse after treatment with at least one anti-cancer therapy, or acombination thereof. In certain other embodiments, there is provided amethod for treating cancer in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of acompound of formula I, or a pharmaceutically acceptable salt, and atherapeutically effective amount of a vinca-alkaloid, or apharmaceutically acceptable salt, wherein the vinca-alkaloid is selectedfrom the group consisting of vincristine, vindesine, vinorelbine andvinblastine, and wherein further the subject is a human who is notundergoing any other anti-cancer treatments; and the subject is (i)refractory to at least one anti-cancer treatment, or (ii) in relapseafter treatment with at least one anti-cancer therapy, or a combinationthereof.

In some embodiments, there is provided a method for treating cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a compound of formula I, or apharmaceutically acceptable salt, and a therapeutically effective amountof a vinca-alkaloid, or a pharmaceutically acceptable salt, wherein: thevinca-alkaloid is selected from the group consisting of vincristine andvinblastine, and the subject is a human who is (i) refractory to atleast one anti-cancer treatment, or (ii) in relapse after treatment withat least one anti-cancer therapy, or a combination thereof. In certainother embodiments, there is provided a method for treating cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a compound of formula I, or apharmaceutically acceptable salt, and a therapeutically effective amountof a vinca-alkaloid, or a pharmaceutically acceptable salt, wherein thevinca-alkaloid is selected from the group consisting of vincristine andvinblastine, and wherein further the subject is a human who is notundergoing any other anti-cancer treatments; and the subject is (i)refractory to at least one anti-cancer treatment, or (ii) in relapseafter treatment with at least one anti-cancer therapy, or a combinationthereof.

In one embodiment, there is provided a method for treating cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a compound of formula I, or apharmaceutically acceptable salt, and a therapeutically effective amountof a vinca-alkaloid, or a pharmaceutically acceptable salt, wherein: thevinca-alkaloid is vincristine, and the subject is a human who is (i)refractory to at least one anti-cancer treatment, or (ii) in relapseafter treatment with at least one anti-cancer therapy, or a combinationthereof. In one other embodiment, there is provided a method fortreating cancer in a subject in need thereof, comprising administeringto the subject a therapeutically effective amount of a compound offormula I, or a pharmaceutically acceptable salt, and a therapeuticallyeffective amount of a vinca-alkaloid, or a pharmaceutically acceptablesalt, wherein the vinca-alkaloid is vincristine, and wherein further thesubject is a human who is not undergoing any other anti-cancertreatments; and the subject is (i) refractory to at least oneanti-cancer treatment, or (ii) in relapse after treatment with at leastone anti-cancer therapy, or a combination thereof.

In one embodiment, there is provided a method for treating cancer in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a compound of formula I, or apharmaceutically acceptable salt, and a therapeutically effective amountof a vinca-alkaloid, or a pharmaceutically acceptable salt, wherein: thevinca-alkaloid is vinblastine, and the subject is a human who is (i)refractory to at least one anti-cancer treatment, or (ii) in relapseafter treatment with at least one anti-cancer therapy, or a combinationthereof. In one other embodiment, there is provided a method fortreating cancer in a subject in need thereof, comprising administeringto the subject a therapeutically effective amount of a compound offormula I, or a pharmaceutically acceptable salt, and a therapeuticallyeffective amount of a vinca-alkaloid, or a pharmaceutically acceptablesalt, wherein the vinca-alkaloid is vinblastine, and wherein further thesubject is a human who is not undergoing any other anti-cancertreatments; and the subject is (i) refractory to at least oneanti-cancer treatment, or (ii) in relapse after treatment with at leastone anti-cancer therapy, or a combination thereof.

In yet other embodiments where the compound of formula I, or apharmaceutically acceptable salt thereof, in combination with avinca-alkaloid, or a pharmaceutically acceptable salt thereof, isadministered as a monotherapy treatment regimen to the subject, thesubject may have a 17p deletion, a TP53 mutation, NOTCH1, a SF3B1mutation, a 11q deletion, or any combination thereof. In one embodimentwhere the compound of formula I, or a pharmaceutically acceptable saltthereof, in combination with a vinca-alkaloid, or a pharmaceuticallyacceptable salt thereof, is administered as a monotherapy treatmentregimen to the subject, the subject has a 17p deletion, a TP53 mutation,or a combination thereof. In another embodiment where the compound offormula I, or a pharmaceutically acceptable salt thereof, in combinationwith a vinca-alkaloid, or a pharmaceutically acceptable salt thereof, isadministered as a monotherapy treatment regimen to the subject, thesubject has NOTCH1, a SF3B1 mutation, a 11q deletion, or any combinationthereof.

Additional Combination Therapies

Provided herein are also methods of treatment in which the compound offormula I, or a pharmaceutically acceptable salt thereof, in combinationwith a vinca-alkaloid, or a pharmaceutically acceptable salt thereof,administered to a subject (e.g., a human) is given to a subject (e.g., ahuman) in additional combination with one or more additional therapies,including one or more of the anti-cancer therapies described above.Thus, in some embodiments, the method for treating cancer in a subject(e.g., a human) in need thereof, comprises administering to the subjecta therapeutically effective amount of a compound of formula I, or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof, in combination with a vinca-alkaloid, or apharmaceutically acceptable salt thereof, together with one or moreadditional therapies, which can be useful for treating the cancer. Theone or more additional therapies may involve the administration of oneor more therapeutic agents.

In some embodiments, the one or more additional therapies involve theuse of a phosphatidylinositol 3-kinase (PI3K) inhibitor, including forexample, Compounds A, B and C, or a pharmaceutically acceptable salt ofsuch compounds.

In other embodiments of the methods described above involving the use ofthe compound of formula I, or a pharmaceutically acceptable saltthereof, in combination with a vinca-alkaloid, or a pharmaceuticallyacceptable salt thereof, in additional combination with one or moreadditional therapies, the one or more additional therapies is other thana therapy using Compound A, Compound B, or Compound C, or apharmaceutically acceptable salt of such compounds. In one embodiment ofthe methods described above involving the use of the compound of formulaI, or a pharmaceutically acceptable salt thereof, in combination with avinca-alkaloid, or a pharmaceutically acceptable salt thereof, inadditional combination with one or more additional therapies, the one ormore additional therapies is other than a therapy using Compound A, or apharmaceutically acceptable salt thereof. In another embodiment of themethods described above involving the use of the compound of formula I,or a pharmaceutically acceptable salt thereof, in combination with avinca-alkaloid, or a pharmaceutically acceptable salt thereof, inadditional combination with one or more additional therapies, the one ormore additional therapies is other than a therapy using Compound B, or apharmaceutically acceptable salt thereof. In yet another embodiment ofthe methods described above involving the use of the compound of formulaI, or a pharmaceutically acceptable salt thereof, in combination with avinca-alkaloid, or a pharmaceutically acceptable salt thereof, inadditional combination with one or more additional therapies, the one ormore additional therapies is other than a therapy using Compound C, or apharmaceutically acceptable salt thereof.

In other embodiments, the one or more additional therapeutic agent maybe an inhibitors of lysyl oxidase-like 2 (LOXL2) and a substance thatbind to LOXL2, including for example, a humanized monoclonal antibody(mAb) with an immunoglobulin IgG4 isotype directed against human LOXL2.

In other embodiments, the one or more additional therapeutic agent maybe an anti-inflammatory agent. Treatment with the one or more additionaltherapeutic agent may be prior to, concomitant with, or followingtreatment with the pharmaceutical composition described herein. In someembodiments, the pharmaceutical composition described herein, iscombined with another therapeutic agent in a single dosage form, whichis then administered prior to, concomitant with or subsequent toadministration with a vinca-alkaloid, or a pharmaceutically acceptablesalt thereof, of this disclosure. Suitable antitumor therapeutics thatmay be used in combination with at least one chemical entity describedherein include, but are not limited to, chemotherapeutic agents, forexample mitomycin C, carboplatin, taxol, cisplatin, paclitaxel,etoposide, doxorubicin, or a combination comprising at least one of theforegoing chemotherapeutic agents. Radiotherapeutic antitumor agents mayalso be used, alone or in combination with chemotherapeutic agents.

The compound of formula I, or a pharmaceutically acceptable saltthereof, can be useful as chemosensitizing agents, and, thus, when usedin combination with a vinca-alkaloid, or a pharmaceutically acceptablesalt thereof, can be useful in combination with other chemotherapeuticdrugs, in particular, drugs that induce apoptosis.

A method for increasing sensitivity of cancer cells to chemotherapy,comprising administering to a subject (e.g., human) undergoingchemotherapy a chemotherapeutic agent together with a compound offormula I, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition thereof, in combination with avinca-alkaloid, or a pharmaceutically acceptable salt thereof, in anamount sufficient to increase the sensitivity of cancer cells to thechemotherapeutic agent is also provided herein. Examples of otherchemotherapeutic drugs that can be used in combination with chemicalentities described herein include topoisomerase I inhibitors(camptothesin or topotecan), topoisomerase II inhibitors (e.g.daunomycin and etoposide), alkylating agents (e.g. cyclophosphamide,melphalan and BCNU), tubulin directed agents (e.g. taxol andvinblastine, or other vinca-alkaloids), and biological agents (e.g.antibodies such as anti CD20 antibody, IDEC 8, immunotoxins, andcytokines). In one embodiment of the method for increasing sensitivityof cancer cells to chemotherapy, the chemotherapeutic agent is otherthan Compound A, or a pharmaceutically acceptable salt thereof. Inanother embodiment of the method for increasing sensitivity of cancercells to chemotherapy, the chemotherapeutic agent is other than CompoundB, or a pharmaceutically acceptable salt thereof. In yet anotherembodiment of the method for increasing sensitivity of cancer cells tochemotherapy, the chemotherapeutic agent is other than Compound C, or apharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula I, or a pharmaceuticallyacceptable salt thereof, or a pharmaceutical composition thereof, isused in combination with Rituxan® (Rituximab) or other agents that workby selectively depleting CD20+ B-cells.

Included herein are methods of treatment in which the compound offormula I, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition thereof, in combination with avinca-alkaloid, or a pharmaceutically acceptable salt thereof, isadministered in combination with an anti-inflammatory agent.Anti-inflammatory agents include but are not limited to NSAIDs,non-specific and COX-2 specific cyclooxgenase enzyme inhibitors, goldcompounds, corticosteroids, methotrexate, tumor necrosis factor receptor(TNF) receptors antagonists, immunosuppressants and methotrexate.Examples of NSAIDs include, but are not limited to ibuprofen,flurbiprofen, naproxen and naproxen sodium, diclofenac, combinations ofdiclofenac sodium and misoprostol, sulindac, oxaprozin, diflunisal,piroxicam, indomethacin, etodolac, fenoprofen calcium, ketoprofen,sodium nabumetone, sulfasalazine, tolmetin sodium, andhydroxychloroquine. Examples of NSAIDs also include COX-2 specificinhibitors (i.e., a compound that inhibits COX-2 with an IC50 that is atleast 50-fold lower than the IC50 for COX-1) such as celecoxib,valdecoxib, lumiracoxib, etoricoxib and/or rofecoxib.

In a further embodiment, the anti-inflammatory agent is a salicylate.Salicylates include but are not limited to acetylsalicylic acid oraspirin, sodium salicylate, and choline and magnesium salicylates. Theanti-inflammatory agent may also be a corticosteroid. For example, thecorticosteroid may be chosen from cortisone, dexamethasone,methylprednisolone, prednisolone, prednisolone sodium phosphate, andprednisone. In some embodiments, the anti-inflammatory therapeutic agentis a gold compound such as gold sodium thiomalate or auranofin. In someembodiments, the anti-inflammatory agent is a metabolic inhibitor suchas a dihydrofolate reductase inhibitor, such as methotrexate or adihydroorotate dehydrogenase inhibitor, such as leflunomide.

In some embodiments, combinations in which at least oneanti-inflammatory compound is an anti-C5 monoclonal antibody (such aseculizumab or pexelizumab), a TNF antagonist, such as entanercept, orinfliximab, which is an anti-TNF alpha monoclonal antibody are used.

In some embodiments, combinations in which at least one therapeuticagent is an immunosuppressant compound such as methotrexate,leflunomide, cyclosporine, tacrolimus, azathioprine, or mycophenolatemofetil are used.

It should be understood that any combinations of the additionaltherapeutic agents described above may be used, as if each and everycombination was individually listed. For example, in certainembodiments, the additional therapeutic agents include a PI3K inhibitorand a LOXL2 inhibitor, such as simtuzumab.

Dosing Regimen and Modes of Administration

In the methods provided herein, the compound of formula I, or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof, is administered in a therapeutically effectiveamount to achieve its intended purpose. As used herein, a“therapeutically effective amount” when referring to the compound ofFormula I is an amount sufficient to modulate Syk expression oractivity, and thereby treat a subject (e.g., a human) suffering anindication, or to ameliorate or alleviate the existing symptoms of theindication. For example, a therapeutically effective amount of thecompound of Formula I or a pharmaceutically acceptable salt thereof maybe an amount sufficient to decrease a symptom of a disease or conditionresponsive to inhibition of Syk activity.

In the methods provided herein, the compound of formula I, or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof, is administered in a therapeutically effectiveamount to achieve its intended purpose. As used herein, a“therapeutically effective amount” when referring to a vinca-alkaloid,or a pharmaceutically acceptable salt thereof, is an amount sufficientto inhibit tubulin growth or formation, or to inhibit or reducemicrotubule formation, or to interfere with spindle formation, andthereby treat a subject (e.g. a human) suffering an indication, or toameliorate or alleviate the existing symptoms of the indication. Forexample, a therapeutically effective amount of a vinca-alkaloid or apharmaceutically acceptable salt thereof may be an amount sufficient todecrease a symptom of a disease or condition responsive to inhibit oftubulin activity and/or formation.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein. In some embodiments, atherapeutically effective amount of the compound of formula I, or apharmaceutically acceptable salt thereof, may (i) reduce the number ofcancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow tosome extent, and preferably stop cancer cell infiltration intoperipheral organs; (iv) inhibit (e.g., slow to some extent andpreferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) delayoccurrence and/or recurrence of a tumor; and/or (vii) relieve to someextent one or more of the symptoms associated with the cancer. Invarious embodiments, the amount is sufficient to ameliorate, palliate,lessen, and/or delay one or more of symptoms of cancer.

The therapeutically effective amount may vary depending on the subject,and disease or condition being treated, the weight and age of thesubject, the severity of the disease or condition, and the manner ofadministering, which can readily be determined by one or ordinary skillin the art.

The dosing regimen of the compound of formula I, or a pharmaceuticallyacceptable salt thereof, in combination with a vinca-alkaloid, or apharmaceutically acceptable salt thereof, in the methods provided hereinmay vary depending upon the indication, route of administration, andseverity of the condition, for example. Depending on the route ofadministration, a suitable dose can be calculated according to bodyweight, body surface area, or organ size. The final dosing regimen isdetermined by the attending physician in view of good medical practice,considering various factors that modify the action of drugs, e.g., thespecific activity of the compound, the identity and severity of thedisease state, the responsiveness of the patient, the age, condition,body weight, sex, and diet of the patient, and the severity of anyinfection. Additional factors that can be taken into account includetime and frequency of administration, drug combinations, reactionsensitivities, and tolerance/response to therapy. Further refinement ofthe doses appropriate for treatment involving any of the formulationsmentioned herein is done routinely by the skilled practitioner withoutundue experimentation, especially in light of the dosing information andassays disclosed, as well as the pharmacokinetic data observed in humanclinical trials. Appropriate doses can be ascertained through use ofestablished assays for determining concentration of the agent in a bodyfluid or other sample together with dose response data.

The formulation and route of administration chosen may be tailored tothe individual subject, the nature of the condition to be treated in thesubject, and generally, the judgment of the attending practitioner. Forexample, the therapeutic index of the compound of formula I, or apharmaceutically acceptable salt thereof, may be enhanced by modifyingor derivatizing the compound for targeted delivery to cancer cellsexpressing a marker that identifies the cells as such. For example, thecompounds can be linked to an antibody that recognizes a marker that isselective or specific for cancer cells, so that the compounds arebrought into the vicinity of the cells to exert their effects locally,as previously described. See e.g., Pietersz et al., Immunol. Rev.,129:57 (1992); Trail et al., Science, 261:212 (1993); andRowlinson-Busza et al., Curr. Opin. Oncol., 4:1142 (1992). A similaranalysis may be applied to treatment with the vinca-alkaloid, or itspharmaceutically acceptable salt, of this disclosure and methods herein.

Dosing Regimen

The therapeutically effective amount of the compound of formula I, or apharmaceutically acceptable salt thereof, may be provided in a singledose or multiple doses to achieve the desired treatment endpoint. Asused herein, “dose” refers to the total amount of an active ingredient(e.g., the compound of formula I, or a pharmaceutically acceptable saltthereof,) to be taken each time by a subject (e.g., a human).

Exemplary doses of the compound of formula I, or a pharmaceuticallyacceptable salt thereof, for a human subject may be between about 0.01mg to about 1800 mg, or between about 0.01 mg to about 1500 mg, orbetween about 10 mg to about 1500 mg, or between about 10 mg to about1300 mg, or between about 10 mg to about 1000 mg, or between about 10 mgto about 800 mg, or between about 10 mg to about 600 mg, or betweenabout 10 mg to about 300 mg, or between about 10 mg to about 200 mg, orbetween about 10 mg to about 100 mg, or between about 100 mg to about800 mg, or between about 100 mg to about 600 mg, or between about 100 mgto about 300 mg, or between about 100 mg to about 200 mg, or betweenabout 200 mg to about 350 mg, or between about 250 mg to about 300 mg,or between about 200 mg to about 400 mg, or between about 400 mg toabout 600 mg, or between about 400 mg to about 800 mg, or between about600 mg or about 800 mg, or between about 800 mg to about 1200 mg, orbetween about 1200 mg to about 1600, or between about 50 mg to about 200mg, or about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125mg, or about 150 mg, or about 175 mg, about 200 mg, about 250 mg, about300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about550 mg, about 600 mg, about 650 mg, about 700 mg, or about 750 mg, orabout 800 mg, or about 850 mg, or about 900 mg, or about 950 mg, orabout 1000 mg, or about 1100 mg, or about 1200 mg, or about 1300 mg, orabout 1400 mg, or about 1500 mg, or about 1600 mg, or about 1800 mg. Inone embodiment, the dose of the compound of formula I, or apharmaceutically acceptable salt thereof, administered to the subject inthe methods provided herein is about 400 mg. In one embodiment, the doseof the compound of formula I, or a pharmaceutically acceptable saltthereof, administered to the subject in the methods provided herein isabout 800 mg.

In other embodiments, the methods provided comprise continuing to treatthe subject (e.g., a human) by administering the doses of the compoundof formula I, or a pharmaceutically acceptable salt thereof, at whichclinical efficacy is achieved or reducing the doses by increments to alevel at which efficacy can be maintained. In a particular embodiment,the methods provided comprise administering to the subject (e.g., ahuman) an initial daily dose of 100 mg to 1000 mg of the compound offormula I, or a pharmaceutically acceptable salt thereof, andadministering subsequent daily doses of the compound of formula I, or apharmaceutically acceptable salt thereof, over at least 6 days, whereineach subsequent daily dose is increased by 50 mg to 400 mg. Thus, itshould also be understood that the dose of the compound of formula I, ora pharmaceutically acceptable salt thereof, may be increased byincrements until clinical efficacy is achieved. Increments of about 25mg, about 50 mg, about 100 mg, or about 125 mg, or about 150 mg, orabout 200 mg, or about 250 mg, or about 300 mg can be used to increasethe dose. The dose can be increased daily, every other day, two, three,four, five or six times per week, or once per week.

The frequency of dosing will depend on the pharmacokinetic parameters ofthe compound administered, the route of administration, and theparticular disease treated. The dose and frequency of dosing may alsodepend on pharmacokinetic and pharmacodynamic, as well as toxicity andtherapeutic efficiency data. For example, pharmacokinetic andpharmacodynamic information about the compound of formula I, or apharmaceutically acceptable salt thereof, can be collected throughpreclinical in vitro and in vivo studies, later confirmed in humansduring the course of clinical trials. Thus, for the compound of formulaI, or a pharmaceutically acceptable salt thereof, used in the methodsprovided herein, a therapeutically effective dose can be estimatedinitially from biochemical and/or cell-based assays. Then, dosage can beformulated in animal models to achieve a desirable circulatingconcentration range that modulates Syk expression or activity. As humanstudies are conducted further information will emerge regarding theappropriate dosage levels and duration of treatment for various diseasesand conditions.

Toxicity and therapeutic efficacy of the compound of formula I, or apharmaceutically acceptable salt thereof, can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe “therapeutic index”, which typically is expressed as the ratioLD₅₀/ED₅₀. Compounds that exhibit large therapeutic indices, i.e., thetoxic dose is substantially higher than the effective dose, arepreferred. The data obtained from such cell culture assays andadditional animal studies can be used in formulating a range of dosagefor human use. The doses of such compounds lies preferably within arange of circulating concentrations that include the ED₅₀ with little orno toxicity.

The administration of the compound of formula I, or a pharmaceuticallyacceptable salt thereof, may be administered under fed conditions. Theterm fed conditions or variations thereof refers to the consumption oruptake of food, in either solid or liquid forms, or calories, in anysuitable form, before or at the same time when the compounds orpharmaceutical compositions thereof are administered. For example, thecompound of formula I, or a pharmaceutically acceptable salt thereof,may be administered to the subject (e.g., a human) within minutes orhours of consuming calories (e.g., a meal). In some embodiments, thecompound of formula I, or a pharmaceutically acceptable salt thereof,may be administered to the subject (e.g., a human) within 5-10 minutes,about 30 minutes, or about 60 minutes consuming calories.

The therapeutically effective amount of the vinca-alkaloid of thisdisclosure, or its pharmaceutically acceptable salt thereof, may beprovided in a single dose or multiple doses to achieve the desiredtreatment endpoint. As used herein, “dose” refers to the total amount ofan active ingredient (e.g., vincristine or vinblastine, for example), ora pharmaceutically acceptable salt thereof,) to be taken each time by asubject (e.g., a human).

Exemplary doses of the vinca-alkaloid of this disclosure, or itspharmaceutically acceptable salt thereof, for a human subject may bebetween about 0.01 mg-M² to about 3.0 mg-M², depending on the identityof the vinca-alkaloid, or between about 0.01 mg-M² to about 2.5 mg-M²,or between about 0.01 mg-M² to about 2.0 mg-M², or between about 0.01mg-M² to about 1.9 mg-M², or between about 0.01 mg-M² to about 1.8mg-M², or between about 0.01 mg-M² to about 1.7 mg-M², or between about0.01 mg-M² to about 1.6 mg-M², or between about 0.01 mg-M² to about 1.5mg-M², or between about 0.01 mg-M² to about 1.4 mg-M², or between about0.01 mg-M² to about 1.3 mg-M², or between about 0.01 mg-M² to about 1.2mg-M², or between about 0.01 mg-M² to about 1.1 mg-M², or between about0.01 mg-M² to about 1.0 mg-M², or between about 0.01 mg-M² to about 0.9mg-M², or between about 0.01 mg-M² to about 0.8 mg-M², or between about0.01 mg-M² to about 0.7 mg-M², or between about 0.01 mg-M² to about 0.6mg-M², or between about 0.01 mg-M² to about 0.5 mg-M², or between about0.01 mg-M² to about 0.45 mg-M², or between about 0.01 mg-M² to about 0.4mg-M², or between about 0.01 mg-M² to about 0.35 mg-M², or between about0.01 mg-M² to about 0.33 mg-M², or between about 0.01 mg-M² to about 0.3mg-M², or between about 0.01 mg-M² to about 0.25 mg-M², or between about0.01 mg-M² to about 0.2 mg-M², or between about 0.01 mg-M² to about 0.15mg-M², or between about 0.01 mg-M² to about 0.01 mg-M², or between about0.1 mg-M² to about 1.8 mg-M², or between about 0.15 mg-M² to about 1.7mg-M², or between about 0.2 mg-M² to about 1.6 mg-M², or between about0.25 mg-M² to about 1.5 mg-M², or between about 0.3 mg-M² to about 1.4mg-M², or between about 0.33 mg-M² to about 1.3 mg-M², or between about0.35 mg-M² to about 1.2 mg-M², or between about 0.4 mg-M² to about 1.1mg-M², or between about 0.45 mg-M² to about 1.0 mg-M², or between about0.5 mg-M² to about 0.9 mg-M², or between about 0.6 mg-M² to about 0.8mg-M². In one embodiment, the dose of the vinca-alkaloid of thisdisclosure, or its pharmaceutically acceptable salt thereof,administered to the subject in the methods provided herein is about 1.5mg-M². In one embodiment, the dose of the vinca-alkaloid of thisdisclosure, or its pharmaceutically acceptable salt thereof,administered to the subject in the methods provided herein is about 1.0mg-M². In one embodiment, the dose of the vinca-alkaloid of thisdisclosure, or its pharmaceutically acceptable salt thereof,administered to the subject in the methods provided herein is about 0.5mg-M².

In other embodiments, the methods provided comprise continuing to treatthe subject (e.g., a human) by administering the doses of thevinca-alkaloid of this disclosure, or a pharmaceutically acceptable saltthereof, at which clinical efficacy is achieved or reducing the doses byincrements to a level at which efficacy can be maintained. The frequencyof dosing will depend on the pharmacokinetic parameters of the compoundadministered, the route of administration, and the particular diseasetreated. The dose and frequency of dosing may also depend onpharmacokinetic and pharmacodynamic, as well as toxicity and therapeuticefficiency data.

Modes of Administration

The pharmaceutical compositions of the compound of formula I may beadministered in either single or multiple doses by any of the acceptedmodes of administration of agents having similar utilities, for exampleas described in those patents and patent applications incorporated byreference, including rectal, buccal, intranasal and transdermal routes,by intra-arterial injection, intravenously, intraperitoneally,parenterally, intramuscularly, subcutaneously, orally, topically, as aninhalant, or via an impregnated or coated device such as a stent, forexample, or an artery-inserted cylindrical polymer.

One mode for administration is parenteral, particularly by injection.The forms in which the compound of formula I, or a pharmaceuticallyacceptable salt thereof, may be incorporated for administration byinjection include aqueous or oil suspensions, or emulsions, with sesameoil, corn oil, cottonseed oil, or peanut oil, as well as elixirs,mannitol, dextrose, or a sterile aqueous solution, and similarpharmaceutical vehicles. Aqueous solutions in saline may alsoconventionally be used for injection. Ethanol, glycerol, propyleneglycol, liquid polyethylene glycol, and the like (and suitable mixturesthereof), cyclodextrin derivatives, and vegetable oils may also beemployed. The proper fluidity can be maintained, for example, by the useof a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

Sterile injectable solutions are prepared by incorporating a compoundaccording to the present disclosure in the required amount in theappropriate solvent with various other ingredients as enumerated above,as required, followed by filtered sterilization. Generally, dispersionsare prepared by incorporating the various sterilized active ingredientsinto a sterile vehicle which contains the basic dispersion medium andthe required other ingredients from those enumerated above. In the caseof sterile powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. In certain embodiments, for parenteral administration,sterile injectable solutions are prepared containing a therapeuticallyeffective amount, e.g., 0.1 to 1000 mg, of the compound of formula I, ora pharmaceutically acceptable salt thereof. It will be understood,however, that the amount of the compound actually administered usuallywill be determined by a physician, in the light of the relevantcircumstances, including the condition to be treated, the chosen routeof administration, the actual compound administered and its relativeactivity, the age, weight, and response of the individual patient, theseverity of the patient's symptoms, and the like.

Oral administration is another route for administration of the compoundof formula I, or a pharmaceutically acceptable salt thereof.Administration may be via capsule or enteric coated tablets, or thelike. In making the pharmaceutical compositions that include thecompound of formula I, or a pharmaceutically acceptable salt thereof,the active ingredient is usually diluted by an excipient and/or enclosedwithin such a carrier that can be in the form of a capsule, sachet,paper or other container. When the excipient serves as a diluent, it canbe in the form of a solid, semi-solid, or liquid material (as above),which acts as a vehicle, carrier or medium for the active ingredient.Thus, the compositions can be in the form of tablets, pills, powders,lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions,syrups, aerosols (as a solid or in a liquid medium), ointmentscontaining, for example, up to 10% by weight of the active compound,soft and hard gelatin capsules, sterile injectable solutions, andsterile packaged powders.

Some examples of suitable excipients in an oral formulation includelactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterilewater, syrup, and methyl cellulose. The formulations can additionallyinclude: lubricating agents such as talc, magnesium stearate, andmineral oil; wetting agents; emulsifying and suspending agents;preserving agents such as methyl and propylhydroxy-benzoates; sweeteningagents; and flavoring agents.

The pharmaceutical compositions of the compound of formula I describedherein can be formulated so as to provide quick, sustained or delayedrelease of the active ingredient after administration to the patient byemploying procedures known in the art. Controlled release drug deliverysystems for oral administration include osmotic pump systems anddissolutional systems containing polymer-coated reservoirs ordrug-polymer matrix formulations. Examples of controlled release systemsare given in U.S. Pat. Nos. 3,845,770; 4,326,525; 4,902,514; and5,616,345. Another formulation for use in the methods of the presentdisclosure employs transdermal delivery devices (patches). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present disclosure in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patches may beconstructed for continuous, pulsatile, or on demand delivery ofpharmaceutical agents.

In some embodiments, the compositions of the compound of formula Idescribed herein are formulated in a unit dosage form. The term “unitdosage forms” refers to physically discrete units suitable as unitarydosages for human subjects, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient (e.g., a tablet, capsule, ampoule).

In other embodiments, the compound of formula I, or a pharmaceuticallyacceptable salt thereof, or a pharmaceutical composition thereof, isadministered orally at a unit dosage of about 50 mg, about 100 mg, about150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about650 mg, about 700 mg, about 800 mg, about 900 mg, about 1100 mg, about1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, or about 1600 mg,about 1700 mg, or about 1800 mg. In other embodiments, the compound offormula I, or a pharmaceutically acceptable salt thereof, isadministered orally at a unit dosage of about 200 mg, about 600 mg, orabout 800 mg, or about 900 mg, or about 1200 mg. In some embodiments,the compound of formula I, or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition thereof, is administered orallyat a unit dosage of about 200 mg, or about 800 mg.

The dosages for oral administration described above for the compound offormula I may be administered once daily or twice daily (BID). Forexample, in certain embodiments, the compound of formula I, or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof, is administered orally at a unit dosage of about 50mg BID, about 100 mg BID, about 150 mg BID, about 200 mg BID, about 250mg BID, about 300 mg BID, about 350 mg BID, about 400 mg BID, about 450mg BID, about 500 mg BID, about 550 mg BID, about 600 mg BID, about 650mg BID, about 700 mg BID, about 800 mg BID, about 900 mg BID, about 1100mg BID, about 1200 mg BID, about 1300 mg BID, about 1400 mg BID, about1500 mg BID, or about 1600 mg BID, about 1700 mg BID, or about 1800 mgBID. In other embodiments, the compound of formula I, or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof, is administered orally at a unit dosage of about200 mg BID, about 400 mg BID, or about 600 mg BID, or about 800 mg BID,or about 1000 mg BID. In some embodiments, the compound of formula I, ora pharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition thereof, is administered orally at a unit dosage of about200 mg BID, or about 800 mg BID. In one embodiment, the compound offormula I, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition thereof, is administered orally at a unitdosage of about 800 mg BID.

The vinca-alkaloid of the disclosure, or the pharmaceutically acceptablesalts thereof, are administered via IV. In one embodiment, thevinca-alkaloid is vincristine sulfate and the amount of the vial is 1mg/1 ml. In some embodiments, the vinca-alkaloid is vincristine sulfateand the vial is 2 ml containing either 1 mg or 2 mg of vincristinesulfate. In another embodiment, “Vincristine Sulfate”, USP is a white tooff-white powder. It is soluble in methanol, freely soluble in water,but only slightly soluble in 95% ethanol. In 98% ethanol, VincristineSulfate, USP has an ultraviolet spectrum with maxima at 221 nm(E+47,100).

“Vincristine Sulfate Injection”, USP is a sterile, preservative-free,single use only solution available for intravenous use in 2 mL (1 mg and2 mg) vials. Each mL contains 1 mg Vincristine Sulfate, USP, 100 mgmannitol and Water for Injection, USP. Q.S. Sulfuric acid or sodiumhydroxide have been added for pH control. The pH of Vincristine SulfateInjection, USP ranges from 4.0 to 5.0.

Articles of Manufacture and Kits

Compositions (including, for example, formulations and unit dosages)comprising the compound of formula I, or a pharmaceutically acceptablesalt thereof, can be prepared and placed in an appropriate container,and labeled for treatment of an indicated condition. Accordingly,provided is also an article of manufacture, such as a containercomprising a unit dosage form of the compound of formula I, or apharmaceutically acceptable salt thereof, and a label containinginstructions for use of the compounds. In some embodiments, the articleof manufacture is a container comprising a unit dosage form of thecompound of formula I, or a pharmaceutically acceptable salt thereof,and at least one pharmaceutically acceptable vehicle. In anotherembodiment, the article of manufacture is a container comprising a unitdosage form of the compound of formula I, or a pharmaceuticallyacceptable salt thereof, and at least one pharmaceutically acceptablevehicle, and a vial containing a vinca-alkaloid, or a pharmaceuticallyacceptable salt.

Kits also are contemplated. For example, a kit can comprise unit dosageforms of the compound of formula I, or a pharmaceutically acceptablesalt thereof, and a package insert containing instructions for use ofthe composition in treatment of a medical condition. In someembodiments, the kits comprises a unit dosage form of the compound offormula I, or a pharmaceutically acceptable salt thereof, and at leastone pharmaceutically acceptable vehicle, and a vial containing asolution of vinca-alkaloid, or a pharmaceutically acceptable saltthereof. The instructions for use in the kit may be for treating acancer, including, for example, a hematologic malignancy or solid tumorcancer malignancy. In some embodiments, the instructions for use in thekit may be for treating cancer, such as leukemia or lymphoma, includingrelapsed and refractory leukemia or lymphoma. In certain embodiments,the instructions for use in the kit may be for treating acutelymphocytic leukemia (ALL), acute myeloid leukemia (AML), chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),myelodysplastic syndrome (MDS), myeloproliferative disease (MPD),chronic myeloid leukemia (CIVIL), multiple myeloma (MM), non-Hodgkin'slymphoma (NHL), indolent non-Hodgkin's lymphoma (iNHL), refractory iNHL,mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldestrom'smacroglobulinemia (WM), T-cell lymphoma, B-cell lymphoma, diffuse largeB-cell lymphoma (DLBCL), lymphoplasmacytic lymphoma (LPL), or marginalzone lymphoma (MZL). In one embodiment, the instructions for use in thekit may be for treating chronic lymphocytic leukemia (CLL) ornon-Hodgkin's lymphoma (NHL). In one embodiment, the NHL is diffuselarge B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicularlymphoma (FL), small lymphocytic lymphoma (SLL), lymphoplasmacyticlymphoma (LPL), and marginal zone lymphoma (MZL). In one embodiment, thehematologic malignancy is indolent non-Hodgkin's lymphoma (iNHL). Incertain embodiments, diseases or conditions indicated on the label caninclude, for example, treatment of cancer.

In certain embodiments of the article of manufacture or the kit, theunit dosage for the compound of formula I has about 50 mg, about 100 mg,about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg,about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg,about 650 mg, about 700 mg, about 800 mg, about 900 mg, about 1100 mg,about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, or about1600 mg, about 1700 mg, or about 1800 mg of the compound of formula I,or a pharmaceutically acceptable salt thereof. In one embodiment, theunit dosage has about 400 mg of the compound of formula I, or apharmaceutically acceptable salt thereof. In one embodiment of thearticle of manufacture or the kit, the unit dosage of the compound offormula I, or a pharmaceutically acceptable salt thereof, is a tablet.

EXAMPLES

The following examples are included to illustrate embodiments of thedisclosure, and are not intended to limit the scope of the disclosure.It should be appreciated by those of skill in the art that thetechniques disclosed herein represent techniques that apply in thepractice of the disclosure. Those of skill in the art would appreciatethat, in light of the present disclosure, changes can be made in theexamples herein without departing from the spirit and scope of thedisclosure.

Example 1 Effect of the Compound of Formula I Compared to Doxorubicin,Vincristine and Cyclophosphamide in 10 Diffuse Large B-Cell Lymphoma(DLBCL), 4 Multiple Myeloma (MM), 2 Follicular Lymphoma (FL), and 1Mantle Cell Lymphoma (MCL) Cell Lines

This Example evaluates the efficacy of the compound of Formula I (FIG.1), or a pharmaceutically acceptable salt thereof, to inhibit malignantB-cell viability in 10 diffuse large B-cell lymphoma (DLBCL), 4 multiplemyeloma (MM), 2 follicular lymphoma (FL), and 1 mantle cell lymphoma(MCL) cell lines. This Example also evaluates and compares the efficacyof doxorubicin, vincristine and cyclophosphamide to inhibit malignantB-cell viability in the same 10 diffuse large B-cell lymphoma (DLBCL), 4multiple myeloma (MM), 2 follicular lymphoma (FL), and 1 mantle celllymphoma (MCL) cell lines, to the efficacy of the compound of formula I,or a pharmaceutically acceptable salt thereof.

Cell Titer Glo Viability Assay:

All procedures were performed at Gilead Sciences, Inc. in Branford, Thecompound of formula (I), cyclophosphamide, doxorubicin and vincristinesulfate were dissolved in DMSO to prepare stock solutions that wereserially diluted three-fold in DMSO in a 96 well plate format at 1000×so the final starting assay concentrations tested would be 10 μM, 200μM, 10 μM, and 1 μM respectively, for each compound. Compound plateswere diluted 1:100 in RPMI without serum or additives creating a 10×stock. Cells were seeded into a 96-well plate at 10,000-20,000 cells perwell in 100 μl of growth media appropriate to the cell line supplementedwith 100 U/L penicillin-streptomycin. OCI-Ly3, OCI-Ly4 and OCI-Ly7 weregrown in Iscove's+20% FBS, OCI-Ly19 was grown in alphaMEM+20% FBS, andall other cell lines were grown in RPMI+10% FBS. To each well, 11 μl of10× compound was added in a final concentration of 0.1% DMSO and cellswere incubated at 37° C. in 5% CO₂ for 72 hours. Cell viability wasassessed with Cell Titer Glo (Promega, Madison, Wis.) following themanufacturer's protocol on an Envision plate reader (Perkin Elmer,Waltham, Mass.). EC₅₀ values were determined using a four parametervariable slope model with GraphPad Prism 6.0 software. All EC₅₀ valuesrepresent mean values of from duplicate dose response curves. Theresults are reported in Table 1 below.

Table 1 summarizes the efficacy of the four compounds tested separatelyin the Cell Titer Glow assay for each respective cell line. The testresults for the compound of formula I and the test results forcyclophosphamide did not show inhibition cell viability in any of thecell lines tested at concentrations <3000 nM and <10000 nM,respectively. Doxorubicin and vincristine EC₅₀ values to inhibit cellviability were calculated and ranged from 3.1-557 nM and <1 nM-103 nMrespectively, as shown in Table 1.

TABLE 1 Inhibition of Viability, EC₅₀ (nM) Compound Vincris- Doxo-Cyclophos- Cell line Disease of Formula I tine rubicin phamide SU-DHL-2DLBCL >3000 65 216 >10000 SU-DHL-4 DLBCL >3000 75 215 >10000 SU-DHL-6DLBCL >3000 12 125 >10000 SU-DHL-10 DLBCL >3000 45 195 >10000 Karpas 422DLBCL >3000 62 305 >10000 OCI-LY3 DLBCL >3000 12 26 >10000 OCI-LY4DLBCL >3000 <1 37 >10000 OCI-LY7 DLBCL >3000 4.6 199 >10000 OCI-LY19DLBCL >3000 1.2 3.1 >10000 Pfeiffer DLBCL >3000 4.0 198 >10000 MinoMCL >3000 42 114 >10000 KMS-11 MM >3000 103 469 >10000 MM1.S MM >3000 5966 >10000 OPM-2 MM >3000 97 557 >10000 RPMI-8266 MM >3000 67 176 >10000WSU-FSCCL FL >3000 58 52 >10000 WSU-NHL FL >3000 39 128 >10000

Example 2 Comparison Effect of the Compound of Formula I in Combinationwith Vincristine Versus Effect of Vincristine Alone in 10 Diffuse LargeB-Cell Lymphoma (DLBCL), 4 Multiple Myeloma (MM), 2 Follicular Lymphoma(FL), and 1 Mantle Cell Lymphoma (MCL) Cell Lines

This Example evaluates the efficacy of the compound of Formula I, or apharmaceutically acceptable salt thereof, in combination withvincristine to inhibit malignant B-cell viability in 10 diffuse largeB-cell lymphoma (DLBCL), 4 multiple myeloma (MM), 2 follicular lymphoma(FL), and 1 mantle cell lymphoma (MCL) cell lines. This Example alsoevaluates and compares the efficacy of vincristine as a single agent toinhibit malignant B-cell viability in the same 10 diffuse large B-celllymphoma (DLBCL), 4 multiple myeloma (MM), 2 follicular lymphoma (FL),and 1 mantle cell lymphoma (MCL) cell lines, to the efficacy of thecompound of formula I, or a pharmaceutically acceptable salt thereof, incombination with vincristine.

Cell Titer Glo Viability Assay:

All procedures were performed at Gilead Sciences, Inc. in Branford,Conn. Vincristine was tested alone as a single agent, or in combinationwith 300 nM of the compound of Formula I, at 4 concentrations within therange of 0.03-10 nM shown to be <EC₅₀ for cell viability as a singleagent. The compound of Formula I and vincristine sulfate were dissolvedin DMSO to prepare stock solutions in separate 96 well plates at 1000×for each compound so the final starting assay concentrations testedwould be 300 nM for the compound of Formula I and 10 nM, 3 nM, 1 nM and0.3 nM for vincristine. Compound plates were diluted and mixed togethersequentially by 1:100 in RPMI without serum or additives creating a 10×stock in 2% DMSO of combinations or single agents. Cells were seededinto a 96-well plate at 10,000-20,000 cells per well in 100 μl of growthmedia appropriate to the cell line supplemented with 100 U/Lpenicillin-streptomycin. OCI-Ly3, OCI-Ly4 and OCI-Ly7 were grown inIscove's+20% FBS, OCI-Ly19 was grown in alphaMEM+20% FBS, and all othercell lines were grown in RPMI+10% FBS. To each well containing cells, 11μl of 10× compound mixture in serum-free media was added in a finalconcentration of 0.2% DMSO and cells were incubated at 37° C. in 5% CO₂for 72 hours. Cell viability was assessed with Cell Titer Glo (CTG,Promega, Madison, Wis.) following the manufacturer's protocol on anEnvision plate reader (Perkin Elmer, Waltham, Mass.). CTG signals wererecorded for individual compound treatments and combinations. Theresults are shown in FIGS. 2A to 2Q.

FIGS. 2A to 2Q detail the inhibition effects of the combination of thecompound of formula I (FIG. 1) and vincristine as compared tovincristine alone in the 17 malignant B-cell lines, representing 4hematological cancer types: DLBCL, MM, FL, and MCL.

Example 3 Comparison Effect of the Combination of the Compound ofFormula I with Vincristine Versus Effect of the Combination of theCompound of Formula I with A) Obretastatin A4, B) Colchicine, C)Doxorubicin, and D) Taxol in the Malignant DLBCL B-Cell Line, DHL-10

This Example evaluates the efficacy of the compound of Formula I (FIG.1), or a pharmaceutically acceptable salt thereof, in combination withvincristine to inhibit malignant B-cell viability in the malignantdiffuse large B-cell lymphoma (DLBCL) cell line DHL-10. This Examplealso evaluates and compares the efficacy of the compound of Formula I,or a pharmaceutically acceptable salt thereof, in combination with A)cobretastatin A4, B) colchicine, C) doxorubicin and D) taxol to inhibitmalignant B-cell viability in the malignant diffuse large B-celllymphoma (DLBCL) cell line DHL-10, to the efficacy of the combination ofthe compound of formula I, or a pharmaceutically acceptable saltthereof, with vincristine.

Cell Titer Glo Viability Assay:

All procedures were performed at Gilead Sciences, Inc. in Branford,Conn. Vincristine, combretastatin A4, colchicine, doxorubicin, and taxolwere dissolved in DMSO to prepare stock solutions that were seriallydiluted three-fold in DMSO in a 96 well plate format at 1000×, so thefinal starting assay concentrations tested would be 1 μM for allcompounds except Doxorubicin which started at 10 μM. Compound plateswere diluted 1:100 in RPMI without serum or additives but containingeither 1% DMSO or 3 μM of the compound of Formula I creating a 10×stock. The DLBCL cell line, DHL-10 was plated at 10,000 cells per wellin RPMI supplemented with 10% FBS and 100 U/L penicillin-streptomycin.To each well containing cells, 11 μl of 10× compound mixture inserum-free media was added in a final concentration of 0.2% DMSO andcells were incubated at 37° C. in 5% CO₂ for 72 hours. Cell viabilitywas assessed using Cell Titer Glo (CTG, Promega, Madison, Wis.)following the manufacturer's protocol on an Envision plate reader(Perkin Elmer, Waltham, Mass.). CTG signals were recorded for individualcompound treatments and combinations. EC₅₀ values were determined usinga four parameter variable slope model with GraphPad Prism 6.0 software.Results are shown in FIG. 2. The shifts in EC₅₀ values for inhibition ofviable DHL-10 cells for the compounds alone or in combination with 300nM of the compound of Formula I are shown in Table 2.

FIGS. 3A to 3C summarize the effect of the combination of the compoundof Formula I and vincristine versus the combination of the compound ofFormula I and combretastatin A4, the compound of Formula I andcolchicine, the compound of Formula I and doxorubicin and the compoundof formula I and taxol.

TABLE 2 Inhibition of cell viability EC₅₀ (nM) No +300 nM CompoundCompound Compound of Formula I of Formula I Fold shift Vincristine 14.13.5 4.0 Colchicine 13.5 14.2 1.0 Combretastatin A4 5.9 4.8 1.2Doxorubicin 152 161 0.9 Taxol 7.1 5.6 1.3

Example 4 Comparison Effect of the Combination of the Compound ofFormula I with Vincristine Versus Effect of the Combination of theCompound of Formula I with Vinblastine in the Malignant DLBCL B-CellLine, DHL-10

This Example evaluates the efficacy of the compound of Formula I, or apharmaceutically acceptable salt thereof, in combination withvincristine to inhibit malignant B-cell viability in the malignantdiffuse large B-cell lymphoma (DLBCL) cell line DHL-10. This Examplealso evaluates and compares the efficacy of the compound of Formula I,or a pharmaceutically acceptable salt thereof, in combination withvinblastine to inhibit malignant B-cell viability in the malignantdiffuse large B-cell lymphoma (DLBCL) cell line DHL-10.

Cell Titer Glo Viability Assay:

Vincristine and vinblastine were tested alone at 3 nM, or in combinationwith 100 nM of the compound of Formula I. The compound of Formula I,vinblastine sulfate and vincristine sulfate were dissolved in DMSO toprepare stock solutions in separate 96 well plates at 1000× for eachcompound so the final starting assay concentrations tested would be 100nM for the compound of Formula I and 3 nM for vinblastine andvincristine. Compound plates were diluted and mixed togethersequentially by 1:100 in RPMI without serum or additives creating a 10×stock in 2% DMSO of combinations or single agents. DHL-10 cells wereseeded into a 96-well plate at 10,000 cells per well in 100 μl of inRPMI+10% FBS supplemented with 100 U/L penicillin-streptomycin. To eachwell containing cells, 11 μl of 10× compound mixture in serum-free mediawas added in a final concentration of 0.2% DMSO and cells were incubatedat 37° C. in 5% CO₂ for 72 hours. Cell viability was assessed using CellTiter Glo (CTG, Promega, Madison, Wis.) following the manufacturer'sprotocol and read on an Envision plate reader (Perkin Elmer, Waltham,Mass.). CTG signals were recorded for individual compound treatment andcombinations. Results are shown in FIG. 4.

FIG. 4 depicts and summarizes the inhibitory effects of the combinationof the compound of FIG. 1 (the compound of Formula I) and one of two ofthe vinca alkaloids, vincristine and vinblastine respectively, in theDLBCL cell line, DHL-10 when compounds were co-administered (FIG. 4).

Example 5 Comparison Effect of the Compound of Formula I in Combinationwith Vincristine in a Syk-Expressing Solid Tumor Cell Line Versus Effectof the Compound of Formula I in Combination with Vincristine in a NonSyk-Expressing Solid Tumor Cell Line

This Example evaluates the efficacy of the compound of Formula I, or apharmaceutically acceptable salt thereof, in combination withvincristine to inhibit cell viability in the Syk-expressing pancreaticcell line, MIA PaCa-2. This Example also evaluates the efficacy of thecombination of the compound of Formula I and vincristine to inhibit cellviability in the non-Syk expressing cell line, HepG2.

Cell Titer Glo Viability Assay:

Vincristine was tested alone, or in combination with 4 concentrations ofthe compound of Formula I (300, 100, 33, and 11 nM), in the malignantpancreatic cell line, MIA PaCa-2 and in the hepatocellular carcinomaHepG2 cell line. The compound of Formula I and vincristine sulfate weredissolved in DMSO to prepare stock solutions in separate 96 well platesat 1000× for each compound so the final starting assay concentrationstested would be 300-11 nM for the compound of Formula I and 3 nM forvincristine. Compound plates were diluted and mixed togethersequentially by 1:100 in RPMI without serum or additives creating a 10×stock in 2% DMSO of combinations or single agents. MIA PaCa-2 and HepG2cells were seeded into 96-well plates at 5,000 cells per well in 100 μlof in RPMI+10% FBS supplemented with 100 U/L penicillin-streptomycin. Toeach well containing cells, 11 μl of 10× compound mixture in serum-freemedia was added in a final concentration of 0.2% DMSO and cells wereincubated at 37° C. in 5% CO₂ for 72 hours. Cell viability was assessedusing Cell Titer Glo (CTG, Promega, Madison, Wis.) following themanufacturer's protocol on an Envision plate reader (Perkin Elmer,Waltham, Mass.). CTG signals were recorded for individual compoundtreatment and combinations. Results are shown in FIGS. 5A and 5B.

FIGS. 5A and 5B summarize the inhibition of cell viability by thecombination of the compound of Formula I and vincristine in theSyk-expressing malignant pancreatic cell line, MiaPaca (FIG. 5A) and inthe non-Syk expressing malignant colon cell line, HepG2 (FIG. 5B).

Syk Protein Assay:

Cell lines were grown logarithmically overnight in RPMI supplementedwith 10% FBS and 100U/L penicillin-streptomycin. 1×10⁷ were collected bycentrifugation at 300×g at room temperature for 8 minutes in 50 mLtubes. Cell pellets were lysed on ice for 15 minutes in 200 uL of 1×RIPAbuffer (Cell Signaling Technology, Danvers Mass.) containing protease(Roche, Palo Alto Calif.) and phosphatase inhibitors (Sigma, Saint LouisMo.; Santa Cruz Technologies, Dallas Tex.). Cells lysates weretransferred to 96-well V-bottom plates and used directly or frozen at−80° C. for use the next day. Proteins were separated with 4-12%SDS-Bis/Tris gels and blotted onto nitrocellulose. Blots were blocked inRockland Odyssey blocking buffer and incubated with a total Sykantibody, 4D10 (Santa Cruz) and pSyk-Y_(525/6) (Cell SignalingTechnologies). The primary antibodies were diluted 1:1000 and incubatedfor 1 hour at room temperature. Blots were washed 3 times 5 minutes inTris-buffered saline containing 1.0% Tween (TBS-T). Blots were thenincubated goat αmouse IgG (H+L), AlexaFluor 680 (Life Sciences, Inc) andGoat αRabbit IgG (H+L), DyLight 800 (Thermo Scientific), each diluted1:20,000 in blocking buffer, for 1 hour at room temperature. Blots werewashed 3 times 5 minutes in TBS-T and analyzed on an Odyssey gel imager(LI-COR).

FIG. 6 depicts the level of Syk expression in the MiaPaca and HepG2malignant colon cell lines (FIG. 6).

Example 6 Determination of Syk Expression in Certain Malignant SolidTumor Cell Lines (FIG. 7)

Syk Protein Assay: Cell lines were grown logarithmically in overnightand 1×10⁷ cells were collected by centrifugation at 300×g at roomtemperature for 8 minutes in 50 mL tubes. Cell pellets were lysed on icefor 15 minutes in 200 uL of 1×RIPA buffer (Cell Signaling Technology,Danvers Mass.) containing protease (Roche, Palo Alto Calif.) andphosphatase inhibitors (Sigma, Saint Louis Mo.; Santa Cruz Technologies,Dallas Tex.). Cells lysates were transferred to 96-well V-bottom platesand used directly or frozen at −80° C. for use the next day. Proteinswere separated with 4-12% SDS-Bis/Tris gels and blotted ontonitrocellulose. Blots were blocked in Rockland Odyssey blocking bufferand incubated with a total Syk antibody, 4D10 (Santa Cruz). The primaryantibody was diluted 1:1000 and incubated for 1 hour at roomtemperature. Blots were washed 3 times 5 minutes in Tris-buffered salinecontaining 1.0% Tween (TBS-T). Blots were then incubated goat αmouse IgG(H+L), AlexaFluor 680 (Life Sciences, Inc), diluted 1:20,000 in blockingbuffer, for 1 hour at room temperature. Blots were washed 3 times 5minutes in TBS-T and analyzed on an Odyssey gel imager (LI-COR). SeeFIG. 7.

Example 7 Efficacy Evaluation of Entospletinib and VincristineIndividually and in Combination in the SU-DHL-10 Mouse Xenograft Modelof Diffuse Large B Cell Lymphoma

Entospletinib was evaluated for efficacy in vivo as a single agent andin combination with vincristine in a subcutaneous cell line tumorxenograft model in male SCID beige mice using the human diffuse largeB-cell lymphoma cell line, SU-DHL-10 in a 2×2 dose level matrix ofEntospletinib and vincristine.

Formulation—Entospletinib was formulated in a complete vehicle of 0.5%HPMC-0.2% Tween 80 (pH 3.5 50 mM acetate buffer, and the lower dose wasprepared by direct dilution). The high dose formulation was mixed usinga stir bar (and Polytroned briefly as needed to break up clumps ofcompound) to form a yellow solution (pH=1.8), which became an opaquesuspension within 30 minutes. The dosing suspension was prepared freshdaily and stored at 4° C. protected from light between treatments.

The formulation for the highest dose group of vincristine was preparedby diluting the stock solution with 0.9% sterile saline, and the lowerdose was prepared by direct dilution of that stock with saline. The highdose formulation had a pH value of 7.08. The dosing formulations wereprepared just prior to each treatment.

Animals—

Male Harlan SCID beige mice (C.B-17/IcrHsd-Prkdc^(scid)Lyst^(bg-J)) wereused in this study. They were 6-7 weeks old on Day 1 of the experimentand were fed irradiated Harlan 2918.15 Rodent Diet and water ad libitum.Animals were housed in static cages with Bed-O'Cobs™ bedding (TheAndersons Lab Bedding Products) inside Biobubble® Clean Rooms thatprovide H.E.P.A filtered air into the bubble environment at 100 completeair changes per hour. All treatments, body weight determinations, andtumor measurements were carried out in the bubble environment. Theenvironment was controlled to a temperature range of 70°±2° F. and ahumidity range of 30-70%.

Cells—

SU-DHL-10 cells were obtained from Leibniz Institute DSMZ-GermanCollection of Microorganisms and Cell Cultures (DSMZ). They were grownin RPMI 1640 medium which was modified with 1% 100 mM Na pyruvate, 1% 1MHEPES buffer, 1% of a 45% glucose solution and supplemented with 10%non-heat-inactivated Fetal Bovine Serum (FBS) and 1% 100×Penicillin/Streptomycin/L-Glutamine (PSG). The growth environment wasmaintained in an incubator with a 5% CO₂ atmosphere at 37° C. The cellswere centrifuged at 200rcf for 8 minutes at 8° C., the supernatantaspirated, and the pellet re-suspended in cold Dulbecco's PhosphateBuffered Saline (DPBS) by pipetting. An aliquot of the homogeneous cellsuspension was diluted with a trypan blue solution and counted using aLuna automated cell counter. The pre-implantation cell viability was91%. The cell suspension was centrifuged at 200rcf for 8 minutes at 8°C. The supernatant was aspirated and the cell pellet was re-suspended incold 50% serum-free medium:50% Matrigel® Matrix (Corning) to generate afinal concentration of 2.5E+07 cells/ml. The cell suspension wasmaintained on wet ice during implantation. Following implantation, analiquot of the remaining cells was diluted with a trypan blue solutionand counted to determine the post-implantation cell viability (91%).Test animals were implanted subcutaneously, high in the axilla (justunder the fore limb) on Day 0 with 5.0×10⁶ cells in 0.2 ml of 50%serum-free medium: 50% Matrigel® Matrix using a 27-gauge needle andsyringe.

All mice were sorted into study groups based on tumor burden (estimatedfrom caliper measurements). Efficacy was evaluated by tumorvolume/weight monitored during and after treatment. Tumor burden (mg)was measured three times weekly by using caliper measurements andconverting to tumor mass by the formula for the volume of a prolateellipsoid assuming unit density as:

Tumor burden (mg)=(L×W ²)/2

-   -   where L and W are the respective orthogonal tumor length and        width measurements (mm). The criteria used for a successful        distribution in this study was a mean tumor burden for all        groups within 10% of the overall mean tumor burden for the study        population Treatment began on Day 16 at an overall mean tumor        burden of 197 mm³.

Group 1: Vehicle control (entospletinib) 0.5% HPMC in 0.2% Tween 80 in50 mM acetate buffer, pH 3.5 was dosed orally every twelve hours for 19days (Days 16-32), and vehicle control (vincristine) 0.9% saline wasdosed intravenously every 7 days, beginning on the third day of oraldosing (Days 18, 25, and 32).

Group 2: Entospletinib was dosed orally at 75 mg/kg every twelve hoursfor 6.5 days (Days 16-22); then lowered to 50 mg/kg every twelve hoursfor 10.5 days (Days 22-32), in combination with saline intravenouslyevery 7 days, beginning on the third day of oral dosing (Days 18, 25,and 32).

Group 3: Entospletinib was dosed orally at 25 mg/kg every twelve hoursfor 19 days (Days 16-32), in combination with saline intravenously every7 days, beginning on the third day of oral dosing (Days 18, 25, and 32).

Group 4: Vehicle for Entospletinib was dosed orally every twelve hoursfor 19 days (Days 16-32), in combination with vincristine at 0.5 mg/kgintravenously every 7 days, beginning on the third day of oral dosing(Days 18, 25, and 32).

Group 5: Vehicle for Entospletinib was dosed orally every twelve hoursfor 19 days (Days 16-32), in combination with vincristine at 0.15 mg/kgintravenously every 7 days, beginning on the third day of oral dosing(Days 18, 25, and 32).

Group 6: Entospletinib was dosed orally at 75 mg/kg every twelve hoursfor 6.5 days (Days 16-22); then lowered due to body weight loss to 50mg/kg every twelve hours for 10.5 days (Days 22-32), in combination withvincristine at 0.5 mg/kg intravenously every 7 days, beginning on thethird day of oral dosing (Days 18, 25, and 32).

Group 7: Entospletinib was dosed orally at 25 mg/kg every twelve hoursfor 19 days (Days 16-32), in combination with vincristine at 0.5 mg/kgintravenously every 7 days, beginning on the third day of oral dosing(Days 18, 25, and 32).

Group 8: Entospletinib was dosed orally at 75 mg/kg every twelve hoursfor 6.5 days (Days 16-22); then lowered to 50 mg/kg every twelve hoursfor 10.5 days (Days 22-32), in combination with vincristine at 0.15mg/kg intravenously every 7 days, beginning on the third day of oraldosing (Days 18, 25, and 32).

Group 9: Entospletinib was dosed orally at 25 mg/kg every twelve hoursfor 19 days (Days 16-32), in combination with vincristine at 0.15 mg/kgintravenously every 7 days, beginning on the third day of oral dosing(Days 18, 25, and 32).

All mice were dosed according to individual body weight on the day oftreatment (0.1 ml/20 g).

-   -   On the 16^(th) day post-tumor implantation, animals with        established tumors (n=9 animals per group) were treated for 3        days by oral gavage with either vehicle or Entospletinib at 75        mg/kg or 25 mg/kg twice daily (BID). On the third day, the        animals received an intravenous dose of saline or vincristine        and plasma and tumor samples were collected 0.5, 2 or 12 hours        post dose. An additional 12 animals per group were treated for        19 days (starting on Day 16 of study) by oral gavage with either        vehicle or Entospletinib at the planned dose levels of 25 mg/kg        and 75 mg/kg BID and intravenous administration of either saline        or vincristine (0.5 mg/kg and 0.15 mg/kg every 7 days (Q7D) for        3 doses starting on the third day after the start of        Entospletinib (Day 19 of study). The 75 mg/kg BID dose level of        Entospletinib was administered for 6.5 days and then reduced to        50 mg/kg BID due to the higher than planned plasma        concentrations measured in plasma samples collected on the third        day of dosing, and weight loss in group being co-treated with        0.5 mg/kg vincristine. This dose level is designated 75/50        mg/kg.

Efficacy was evaluated by tumor volume measurements. Plasma levels ofEntospletinib and vincristine were evaluated on the third day of dosingEntospletinib (Day 19) and the last day of dosing (Day 32). Tolerabilitywas evaluated by daily observations, body weight (3 times per week) andcirculating blood cell counts at the end of the study.

All dose levels and combinations evaluated were tolerated, following the75 mg/kg entospletinib dose reduction to 50 mg/kg. The doses of 25 and50 mg/kg BID Entospletinib resulted in plasma concentrations at the endof study that exceed the in vitro pervanadate-stimulated mouse wholeblood EC₅₀ and EC₅₀ values for inhibition of SYK, respectively, atC_(min) (trough). Vincristine administered at both 0.15 and 0.5 mg/kgresulted in significant inhibition of tumor growth. Entospletinib dosedalone at 25 or 75/50 mg/kg inhibited tumor growth but to a lesser degreethan that seen with vincristine.

The addition of Entospletinib at the 75/50 mg/kg level resulted insignificant improvement in tumor growth inhibition over vincristinealone with an increase in % tumor growth inhibition (TGI) from 85% to96% for 0.5 mg/kg vincristine and an increase in % TGI form 42% to 71%for 0.15 mg/kg vincristine. The addition of 25 mg/kg Entospletinib toeither dose of vincristine did not significantly increase the tumorgrowth inhibition. While the groups receiving either Entospletinib orvincristine as single agents had no complete or partial regressions, 50%of the mice receiving the combination of 75/50 mg/kg Entospletinib with0.5 mg/kg vincristine had partial responses and 8% had completeregression and 8% were tumor free at the end of study. The tumor growthinhibition and tolerability of Entospletinib and vincristine alone andin combination are summarized in the table below.

Summary of Efficacy with Entospletinib and Vincristine at ToleratedDoses

Day 31% Treatment Tumor Growth (Group number) Inhibition^(a)Significance (P value) Vehicle for ENTO BID + NA^(b) Vehicle for VCRQ7Dx3 (1) ENTO 75/50 mg/kg BID + 39 <0.00001 vs. vehicle Vehicle for VCRQ7Dx3 (2) ENTO 25 mg/kg BID + 20 0.0455 vs. vehicle Vehicle for VCRQ7Dx3 (3) Vehicle for ENTO + 85 <0.00001 vs. vehicle VCR 0.5 mg/kg Q7Dx3(4) Vehicle for ENTO + 42 <0.00001 vs. vehicle VCR 0.15 mg/kg Q7Dx3 (5)ENTO 75/50 mg/kg BID + 96 0.001 vs. VCR 0.5 VCR 0.5 mg/kg Q7Dx3 (6)mg/kg ENTO 25 mg/kg BID + 90 0.3866 vs. VCR 0.5 VCR 0.5 mg/kg Q7Dx3 (7)mg/kg ENTO 75/50 mg/kg BID + 71 <0.00001 vs. VCR 0.15 VCR 0.15 mg/kgQ7Dx3 (8) mg/kg ENTO 25 mg/kg BID + 58 0.0754 vs. VCR 0.15 VCR 0.15mg/kg Q7Dx3 (9) mg/kg ^(a)The percentage of tumor growth inhibition (%TGI) was calculated using the delta of the mean treated tumor valuesdivided by the delta of the mean control tumor values expressed as apercentage and then subtracted from 100%. ^(b)NA does not apply sincethis is the control

The administration of combinations of Entospletinib with vincristine toanimals baring the SU-DHL-10 subcutaneous xenograft demonstrated that adose of entospletinib which results in a calculated EC₅₀ trough coverageof SYK resulted in significant increase in tumor growth inhibition aswell as an increase to 50% partial tumor regressions compared to 0% withentospletinib or vincristine treatment alone, with 8% completeregression.

What is claimed is:
 1. A method for treating cancer in a human in needthereof, comprising administering to the human a therapeuticallyeffective amount of a compound of formula I:

or a pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of a vinca-alkaloid, or a pharmaceutically acceptablesalt.
 2. The method of claim 1, wherein the vinca-alkaloid is selectedfrom the group consisting of vincristine, vinblastine, vindesine,vinorelbine, desoxyvincaminol, vincaminol, vinburnine, vincamajine, andvineridine.
 3. The method of claim 2, wherein the vinca-alkaloid isselected from the group consisting of vincristine, vinblastine,vindesine, and vinorelbine.
 4. The method of claim 1 wherein thevinca-alkalod is vincristine.
 5. The method of claim 1 wherein thevinca-alkalod is vinblastine.
 6. The method of claim 1, wherein thecompound of formula (I) is present in a pharmaceutical compositioncomprising the compound of formula (I), and at least onepharmaceutically acceptable excipient
 7. The method of claim 1, whereinthe compound of formula (I) or a pharmaceutically acceptable saltthereof is administered before the vinca-alkaloid, or a pharmaceuticallyacceptable salt thereof.
 8. The method of claim 1, wherein the vincaalkaloid, or a pharmaceutically acceptable salt thereof, is administeredbefore the compound of formula (I) or a pharmaceutically acceptable saltthereof.
 9. The method of claim 1, wherein the compound of formula (I)or a pharmaceutically acceptable salt thereof, and the vinca-alkaloid,or a pharmaceutically acceptable salt thereof, are administeredsimultaneously.
 10. The method of claim 1, wherein each of the compoundof formula I and the vinca-alkaloid, or pharmaceutically acceptablesalts thereof, is independently administered, wherein the compound offormula I is administered twice a day and wherein further thevinca-alkaloid is administered once a week.
 11. The method of claim 1,wherein the compound of formula (I) or a pharmaceutically acceptablesalt thereof is administered at a dose between 100 mg and 800 mg and thevinca-alkaloid, or a pharmaceutically acceptable salt thereof, isadministered at a dose between about 0.1 mg-M² and 1.5 mg-M².
 12. Themethod of claim 1, wherein the compound of formula (I) or apharmaceutically acceptable salt thereof is administered at a dosebetween 200 mg and 400 mg and the vinca-alkaloid or a pharmaceuticallyacceptable salt thereof is administered at a dose between 0.25 mg-M² and1.0 mg-M², wherein the vinca-alkaloid is selected from the groupconsisting of vincristine and vinblastine.
 13. The method of claim 1,wherein the human who has cancer is (i) refractory to at least onechemotherapy treatment, or (ii) is in relapse after treatment withchemotherapy, or a combination thereof.
 14. The method of claim 1,wherein the human has not previously been treated for the cancer. 15.The method of claim 1, wherein the human has a 17p deletion, a TP53mutation, or a combination thereof.
 16. The method of claim 15, whereinthe human further has NOTCH1, a SF3B1 mutation, a 11q deletion, or anycombination thereof.
 17. The method of claim 1 wherein: thevinca-alkaloid is selected from the group consisting of vincristine andvinblastine, and the human is (i) refractory to at least one anti-cancertreatment, or (ii) in relapse after treatment with at least oneanti-cancer therapy, or a combination thereof.
 18. The method of claim17 wherein the human is not undergoing any other anti-cancer treatments.19. The method of claim 17, wherein the human is not undergoing anyother anti-cancer treatments using one or more PI3K inhibitors.
 20. Themethod of claim 18, wherein the human is refractory to at least oneanti-cancer treatment.
 21. The method of claim 18, wherein the human isin relapse after treatment with at least one anti-cancer treatment. 22.The method of claim 18, wherein about 200 mg to about 800 mg of thecompound of formula I, or a pharmaceutically acceptable salt thereof, isadministered to the human twice daily.
 23. The method of claim 22,wherein 100-400 mg of the compound of formula I, or a pharmaceuticallyacceptable salt thereof, is administered to the human twice daily. 24.The method of claim 18, wherein the human has a 17p deletion, a TP53mutation, or a combination thereof and about 400 mg of the compound offormula I, or a pharmaceutically acceptable salt thereof, isadministered to the human twice daily.
 25. The method of claim 1,wherein the cancer is a hematologic malignancy.
 26. The method of claim1, wherein the cancer is a leukemia.
 27. The method of claim 1, whereinthe leukemia is chronic lymphocytic leukemia (CLL).
 28. The method ofclaim 1, wherein the cancer is a lymphoma.
 29. The method of claim 1,wherein the lymphoma is non-Hodgkin's lymphoma (NHL).
 30. The method ofclaim 29, wherein the NHL is diffuse large B-cell lymphoma (DLBCL),mantle cell lymphoma (MCL), follicular lymphoma (FL), small lymphocyticlymphoma (SLL), lymphoplasmacytic lymphoma (LPL), and marginal zonelymphoma (MZL).
 31. The method of claim 1, wherein the cancer isselected from the group consisting of acute lymphocytic leukemia (ALL),acute myeloid leukemia (AML), Burkitt's lymphoma (BL), chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),myelodysplastic syndrome (MDS), myeloproliferative disease (MPD),chronic myeloid leukemia (CIVIL), multiple myeloma (MM), non-Hodgkin'slymphoma (NHL), indolent non-Hodgkin's lymphoma (iNHL), refractory iNHL,mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldestrom'smacroglobulinemia (WM), T-cell lymphoma, B-cell lymphoma, Hodgkin'slymphoma, diffuse large B-cell lymphoma (DLBCL), lymphoplasmacyticlymphoma (LPL), and marginal zone lymphoma (MZL).
 32. The method ofclaim 1, wherein the cancer is a solid tumor.
 33. The method of claim 1,wherein the cancer is a solid tumor and expresses spleen tyrosine kinase(Syk) activity.
 34. The method of claim 32, wherein the solid tumorcancer is selected from the group consisting of pancreatic, lung,colorectal cancer, ovarian, breast, esophageal, adenocarcinoma andhepatocellular.
 35. The method of claim 1 wherein each of the compoundof formula I and the vinca-alkaloid, or pharmaceutically acceptablesalts thereof, is independently administered, wherein the compound offormula I is administered once.
 36. The method of claim 1 wherein eachof the compound of formula I and the vinca-alkaloid, or pharmaceuticallyacceptable salts thereof, is independently administered, wherein thecompound of formula I is administered once a day and wherein further thevinca-alkaloid is administered once a week.